Method for producing a cell culture substrate

ABSTRACT

A main object of the invention is to provide a new method for producing a cell culture substrate used to cause cells to adhere in a highly precise form onto a base material and then culture the cells. To attain the object, the invention provides a method for producing a cell culture substrate comprising: a patterning substrate forming process of forming, on a base material, a cell culture patterning layer wherein a cell adhesion portion having cell adhesive properties and a cell adhesion-inhibiting portion having cell adhesion-inhibiting properties can be formed by action of a photocatalyst by energy irradiation, thereby forming a patterning substrate; an energy irradiating process of irradiating the energy onto the cell culture patterning layer, thereby forming the cell adhesion portion and the cell adhesion-inhibiting portion in a pattern form by action of the photocatalyst; and a cell-containing liquid applying process of applying a cell-containing liquid onto the cell adhesion portion by a region-selecting applying method of applying the cell-containing liquid selectively onto the patterned cell adhesion portion.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for producing a cell culturesubstrate on which cells are caused to adhere in a highly precisepattern form.

2. Description of the Related Art

At present, cell cultures of various animals and plants are performed,and also new cell culture methods are in development. The technologiesof the cell culture are utilized, such as, to elucidate the biochemicalphenomena and natures of cells and to produce useful substances.Furthermore, with cultured cells, an attempt to investigate thephysiological activity and toxicity of artificially synthesized medicalsis under way.

Some cells, particularly a lot of animal cells have the adhesiondependency of adhering to some materials and growing thereon, and cannotsurvive for a long period under a flotation condition out of organisms.For culturing cells having such adhesion dependency, a carrier to whichcells can adhere is necessary, and in general, a plastic culturing platewith uniformly applied cell adhesive proteins such as collagen,fibronectin and the like is used. It is known that these cell adhesiveproteins act on cultured cells, make the cells adhere easily, and exertan influence on the form of cells.

On the other hand, there is a technology reported of adhering culturedcells only onto a small part on a base material and arranging them. Bysuch a technology, it is made possible to apply cultured cells toartificial organs, biosensors, bioreactors and the like. As the methodof arranging cultured cells, there is a method adopted in which a basematerial having a surface that forms a pattern different in easiness ofadhesion to cells is used, cells are cultured on the surface of thisbase material and allowed to adhere only onto surfaces processed so thatcells adhere, and thereby the cells are arranged.

For example, in Japanese Patent Application Laid-Open (JP-A) No.2-245181, an electric charge-retaining medium on which an electrostaticpattern is formed is applied to culture cells for the purpose ofproliferating nerve cells in a form of circuit, and the like.Furthermore, JP-A No. 3-7576 tries to arrange cultured cells on asurface on which a cell adhesion-inhibiting or cell adhesivephotosensitive hydrophilic polymer has been patterned by aphotolithography method.

Furthermore, JP-A No. 5-176753 discloses a cell culture base material onwhich a substance such as collagen and the like affecting on theadhesion ratio and form of cells is patterned, and a method of producingthis base material by a photolithography method. By culturing cells onsuch a base material, a larger amount of cells can be adhered on asurface on which collagen or the like is patterned, to realizepatterning of cells.

However, such patterning of cell culture regions may be required to behighly precise depending on applications. In the case of conductingpatterning by a photolithography method using a photosensitive materialas described above, a highly precise pattern can be obtained; however, acell adhesive material is required to have photosensitivity, and it isdifficult in many cases to conduct chemical modification to impart suchphotosensitivity to, for instance, biopolymers and the like; therebyleading to extremely narrow width in selectivity of cell adhesivematerials, problematically. Furthermore, in a photolithography methodusing a photo resist, it is necessary to use a liquid developer and thelike, and these can affect adversely in culturing cells in some cases.

Furthermore, as a method of forming a highly precise pattern of a celladhesive material, a Micro Contact Printing method is proposed by GeorgeM. Whitesides, Harvard University (for example, U.S. Pat. Nos. 5,512,131and 5,900,160, JP-A Nos. 9-240125 and 10-12545 etc). However, there is aproblem in that it is difficult to industrially produce a cell culturebase material having a pattern of a cell adhesive material using thismethod.

SUMMARY OF THE INVENTION

Thus, it has been desired to provide a new method for producing a cellculture substrate used to cause cells to adhere in a highly precisepattern onto a base material and then culture the cells.

The present invention provides a method for producing a cell culturesubstrate, comprising: a patterning substrate forming process offorming, on a base material, a cell culture patterning layer wherein acell adhesion portion having cell adhesive properties and a celladhesion-inhibiting portion having cell adhesion-inhibiting propertiescan be formed by an action of a photocatalyst by energy irradiation,thereby forming a patterning substrate; an energy irradiating process ofirradiating an energy onto the cell culture patterning layer, therebyforming the cell adhesion portion and the cell adhesion-inhibitingportion in a pattern form by the action of the photocatalyst; and acell-containing liquid applying process of applying a cell-containingliquid onto the cell adhesion portion by a region-selecting applyingmethod of applying the cell-containing liquid selectively onto the celladhesion portion in the patterned form.

In the present invention, a cell-containing liquid is applied onto thecell adhesion portion, which is formed in a pattern form by action of aphotocatalyst, by a region-selecting applying method of applying thecell-containing liquid selectively, such as ink-jetting method;therefore, it is possible to produce a cell culture substrate whereinthe adhesion pattern of cells is highly precise. For example, in thecase of forming cellar tissues or the like, it is necessary to cause twoor more kinds of cells to adhere onto a single substrate; according tothe present invention, two or more kinds of cells can be extremelyeasily caused to adhere onto a single substrate.

The conventionally-performed method of inoculating cells onto the wholesurface of a base material and then patterning the cells is ordinarily amethod of inoculating more cells than cells to be actually patternedonto the whole and then washing off the cells which are left overwithout being patterned. Therefore, loss is generated out of the usedcells. For this reason, it is difficult to apply such a patterningmethod to the case of the kind of cells which cannot be easily obtained.On the other hand, the present invention has the following advantagesthat: a loss of the number of cells can be decreased since thecell-containing liquid is applied only onto a target region; and thusvarious kinds of cells can be patterned.

The method of the invention for producing a cell culture substrate canbe classified into 6 embodiments, dependently on difference in the kindof the patterning substrate in the patterning substrate forming processand in the energy irradiating process.

The method of the first embodiment for producing a cell culturesubstrate is a method wherein: the above-mentioned patterning substratecomprises a base material, and a cell culture patterning layer which isformed on the base material and has a photocatalyst-containing celladhesive layer comprising at least a photocatalyst and a cell adhesivematerial which has cell adhesive properties and is decomposed ordenatured by action of the photocatalyst on the basis of energyirradiation; and the energy irradiating process is a process ofirradiating the energy from a given direction, thereby forming a patterncomposed of a cell adhesion-inhibiting portion where the cell adhesivematerial comprised in the photocatalyst-containing cell adhesive layeris decomposed or denatured, and a cell adhesion portion which is otherthan the cell adhesion-inhibiting portion.

The method of the second embodiment for producing a cell culturesubstrate is a method wherein: the above-mentioned patterning substratecomprises a base material, and a cell culture patterning layer which isformed on the base material and has a photocatalyst-containing celladhesion-inhibiting material layer comprising at least a photocatalystand a cell adhesion-inhibiting material which has celladhesion-inhibiting properties of inhibiting adhesion onto cells and isdecomposed or denatured by action of the photocatalyst on the basis ofenergy irradiation; and the energy irradiating process is a process ofirradiating the energy from a given direction, thereby forming a patterncomposed of a cell adhesion portion where the cell adhesion-inhibitingmaterial comprised in the photocatalyst-containing celladhesion-inhibiting material layer is decomposed or denatured, and acell adhesion-inhibiting portion which is other than the cell adhesionportion.

In the first and second embodiments, their cell culture patterninglayers have the photocatalyst-containing cell adhesive layer and thephotocatalyst-containing cell adhesion-inhibiting material layer,respectively, which each comprise the photocatalyst; therefore, the cellculture patterning layer can be rendered a single layer. Thus, theembodiments are advantageous from the viewpoint of process, and give agood patterning sensitivity.

The method of the third embodiment for producing a cell culturesubstrate is a method wherein: the patterning substrate comprises a basematerial, and a cell culture patterning layer which is formed on thebase material and has a photocatalyst-containing layer comprising atleast a photocatalyst and a cell adhesive layer formed on thephotocatalyst-containing layer; the cell adhesive layer at leastcomprises a cell adhesive material which has cell adhesive propertiesand is decomposed or denatured by action of the photocatalyst on thebasis of energy irradiation; and the energy irradiating process is aprocess of irradiating the energy from a given direction, therebyforming a pattern composed of a cell adhesion-inhibiting portion wherethe cell adhesive material comprised in the cell adhesive layer isdecomposed or denatured, and a cell adhesion portion which is other thanthe cell adhesion-inhibiting portion.

The method of the fourth embodiment for producing a cell culturesubstrate is a method wherein: the above-mentioned patterning substratecomprises a base material, and a cell culture patterning layer which isformed on the base material and has a photocatalyst-containing layercomprising at least a photocatalyst and a cell adhesion-inhibitingmaterial layer formed on the photocatalyst-containing layer; celladhesion-inhibiting material layer comprises a cell adhesion-inhibitingmaterial which has cell adhesion-inhibiting properties of inhibitingadhesion onto the cells and is decomposed or denatured by action of thephotocatalyst on the basis of energy irradiation; and the energyirradiating process is a process of irradiating the energy from a givendirection, thereby forming a pattern composed of a cell adhesion portionwhere the cell adhesion-inhibiting material comprised in the celladhesion-inhibiting material layer is decomposed or denatured and a celladhesion-inhibiting portion which is other than the cell adhesionportion.

In the third and fourth embodiments, the cell adhesive layer and thecell adhesion-inhibiting material layer are formed on thephotocatalyst-containing layer; accordingly, the possibility that cellsdirectly contact the photocatalyst is small. For this reason, theembodiments have an advantage that a fear that cells deteriorates withthe passage of time is decreased. Furthermore, these embodiments alsohave an advantage that the embodiments can be carried out even if thephotocatalyst material cannot be mixed directly with the cell adhesivematerial or cell adhesion-inhibiting material.

The method of the fifth embodiment for producing a cell culturesubstrate is a method wherein: the above-mentioned patterning substratecomprises a base material and a cell culture patterning layer which isformed on the base material and has a cell adhesive layer comprising acell adhesive material that has cell adhesive properties and isdecomposed or denatured by action of a photocatalyst on the basis ofenergy irradiation; and the energy irradiating process is a process ofarranging the cell adhesive layer and a photocatalyst-containing layerside substrate having a base body and a photocatalyst-containing layercomprising the photocatalyst to dispose the cell adhesive layer and thephotocatalyst-containing layer facing each other, and then irradiatingthe energy onto the resultant from a given direction, thereby forming apattern composed of a cell adhesion-inhibiting portion where the celladhesive material comprised in the cell adhesive layer is decomposed ordenatured, and a cell adhesion portion which is other than the celladhesion-inhibiting portion.

The method of the sixth embodiment for producing a cell culturesubstrate is a method wherein: the above-mentioned patterning substratecomprises a base material and a cell culture patterning layer which isformed on the base material and has a cell adhesion-inhibiting materiallayer comprising a cell adhesion-inhibiting material that has celladhesion-inhibiting properties of inhibiting adhesion onto cells and isdenatured by action of a photocatalyst on the basis of energyirradiation; and the energy irradiating process is a process ofarranging the cell adhesion-inhibiting material layer and aphotocatalyst-containing layer side substrate having a base body and aphotocatalyst-containing layer comprising the photocatalyst to disposethe cell adhesion-inhibiting material layer and thephotocatalyst-containing layer facing each other, and then irradiatingthe energy onto the resultant from a given direction, thereby forming apattern composed of a cell adhesion portion where the celladhesion-inhibiting material comprised in the cell adhesion-inhibitingmaterial layer is decomposed or denatured, and a celladhesion-inhibiting portion which is other than the cell adhesionportion.

The fifth and sixth embodiments have an advantage that the cell culturesubstrate itself does not need to contain any photocatalyst and thus thepossibility that cells deteriorate with the passage of time is small.

In the methods of the second, fourth and sixth embodiments for producinga cell culture substrate of the present invention, it is preferred thatthe cell adhesion portion is a hydrophilic region and the celladhesion-inhibiting portion is a water-repellent region for thefollowing reasons. In the case of applying a cell-containing liquid byuse of the region-selecting applying method of applying thecell-containing liquid selectively, such as ink-jetting method, thecell-containing liquid can be highly precisely applied onto the celladhesion portion on the basis of the matter that the cell adhesionportion is hydrophilic and the cell adhesion-inhibiting portion is waterrepellent since the cell-containing liquid is a hydrophilic material oran aqueous solution. Also, when cells are caused to adhere onto theboundary region between hydrophilic and water repellent regions, whichhave different natures, a change in the form of the cells, such asextension or orientation, may be caused in accordance with the kind ofthe cells. According to a report, such a change promotes theorganization of the cells. According to the above-mentioned methods,such a change in the form of cells can be promoted as the case may be(see, for example, Donald E. Ingber et al., Journal of Cell Biology(1989), pp. 317-, and Kevin E. Healy, et al., Biotechnology andBioengineering (1994), pp. 792-).

In the method of the invention for producing a cell culture substrate,light-shielding portions may be formed on the base material or thephotocatalyst-containing layer in a pattern form. When thelight-shielding portions are formed in this way, troublesome steps, suchas a positioning step, are unnecessary since it is unnecessary to useany photomask at the time of energy irradiation. Moreover, a more highlyprecise pattern can be obtained in this case than in the case of using aphotomask or the like since the distance between the cell culturepatterning layer and the light-shielding portions are extremely small.

Furthermore, in the production method of the invention, it is preferredthat the region-selecting applying method is ink-jetting method.According to this, the production can be made effective.

The present invention also provides an ink-jetting cell-containingliquid comprising cells, a culture solution and a biological celladhesive material, and jets out in a pattern form onto a substrate byink-jetting method, thereby forming a cell culture substrate onto whichthe cells adhere in the pattern form. This ink-jetting cell-containingliquid is good in adhesive property to the substrate. In general,biological cell adhesive materials are high in viscosity. Byincorporating such a biological cell adhesive material into thecell-containing liquid, the viscosity of the cell-containing liquid canbe raised to prevent the cells jetted out from moving around in thecell-containing liquid.

The present invention also provides an ink-jetting cell-containingliquid jets out in a pattern form onto a substrate by ink-jetting methodto form a cell culture substrate onto which cells adhere in the patternform, and the cells being micro-encapsulated. Since the cells aremicro-encapsulated, the cells are not damaged, for example, when thecell-containing liquid is jetted out. As a result, a good-quality cellculture substrate can be produced.

These ink-jetting cell-containing liquids are preferably used when theregion-selecting applying method is ink-jetting method in the method ofthe invention for producing a cell patterning substrate. The ink-jettingmethod referred to herein is a method of jetting out cells or a cellliquid. The system for the ink-jetting may be selected at will if thesystem does not give any damage to cells. Examples thereof includepiezoelectrically driving, bubble jetting (registered trade mark),electrostatic jetting, electric field jetting, ultrasonic jetting,centrifugal jetting, and inertial force head systems. Thepiezoelectrically driving system is particularly preferred since thesystem does not give any damage to cells.

The present invention also provides an ink-jetting device for producinga cell culture substrate comprising a cell-containing liquid supplyingsection into which a cell-containing liquid is filled, a jetting-outsection having a piezoelectrically driving head unit for jetting out thecell-containing liquid supplied from the cell-containing liquidsupplying section onto a cell culture patterning substrate, and a stagefor fixing the cell culture patterning substrate, wherein thecell-containing liquid supplying section is provided with a stirringmeans for dispersing cells homogeneously in the cell-containing liquidfilled, and the jetting-out section and the stage are provided with oneor more temperature controlling means for keeping the temperature of thecell-containing liquid constant before and after the jetting-out. By useof this ink-jetting device for producing a cell culture substrate, ahigh-quality cell culture substrate can be produced.

This device is preferably provided with a humidity controlling means forcontrolling the humidity between the piezoelectrically driving head unitand the cell culture patterning substrate fixed onto the stage. Thedevice makes it possible to prevent the extinction or damage of cellsbased on the drying of the cell-containing liquid, a drop in activity ofthe cells, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are process charts illustrating an example of the energyirradiating process in the method of the invention for producing a cellculture substrate.

FIGS. 2A and 2B are process charts illustrating another example of theenergy irradiating process in the method of the invention for producinga cell culture substrate.

FIG. 3 is a schematic sectional view showing an example of thephotocatalyst-containing layer side substrate used in the invention.

FIG. 4 is a schematic sectional view showing another example of thephotocatalyst-containing layer side substrate used in the invention.

FIG. 5 is a schematic sectional view showing still another example ofthe photocatalyst-containing layer side substrate used in the invention.

FIG. 6 is a schematic sectional view illustrating an ink-jetting devicefor producing a cell culture substrate of the invention.

FIGS. 7A and 7B are each a schematic sectional view for explaining abase material used in the method of the invention for producing a cellculture substrate.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a cell culture substrate producingmethod which can give a cell culture substrate onto which cells adherein a highly precise pattern form; an ink-jetting cell-containing liquid(i.e., a cell-containing liquid for ink-jet) which is preferably used toproduce a cell culture substrate by ink-jetting method; and anink-jetting device for producing effectively a cell culture substratehaving a high quality. The following will describe them separately.

A. Method for Producing a Cell Culture Substrate

First, the method of the invention for producing a cell culturesubstrate is described. The method is a method for producing a cellculture substrate, comprising: a patterning substrate forming process offorming, on a base material, a cell culture patterning layer wherein acell adhesion portion having cell adhesive properties and a celladhesion-inhibiting portion having cell adhesion-inhibiting propertiescan be formed by action of a photocatalyst by energy irradiation,thereby forming a patterning substrate; an energy irradiating process ofirradiating the energy onto the cell culture patterning layer, therebyforming the cell adhesion portion and the cell adhesion-inhibitingportion in a pattern form by action of the photocatalyst; and acell-containing liquid applying process of applying a cell-containingliquid onto the cell adhesion portion by a region-selecting applyingmethod of applying the cell-containing liquid selectively onto thepatterned cell adhesion portion.

In the method of the invention for producing a cell culture substrate, acell-containing liquid is applied onto the cell adhesion portion by theregion-selecting applying method capable of applying the liquidselectively; therefore, a cell culture substrate having a highly precisepattern can be produced. Since such an applying method is used, pluralkinds of cell-containing liquids can be applied. Accordingly, thepresent method can be effectively used to form, for example, cellartissues composed of plural kinds of cells.

When cells are caused to adhere onto a cell culture region and culturedto form tissues, it is generally necessary that individuals of the cellsare subjected to form-change, and arranged. In the case of applying acell-containing liquid onto an ordinary base material, it is difficultto cause this form-change or arrangement and thus form tissues. On theother hand, according to the invention, a cell-containing liquid isapplied onto the cell adhesion portion of the cell culture patterninglayer, which has the cell adhesion portion and the celladhesion-inhibiting portion. It is possible that the cells applied ontothe cell adhesion portion are easily arranged or subjected toform-change along the pattern composed of the cell adhesion portion andthe cell adhesion-inhibiting portion. Thus, the present method has anadvantage that tissues are easily formed.

The following will describe each of the processes in the method of theinvention for producing a cell culture substrate.

1. Patterning Substrate Forming Process

The patterning substrate forming process in the invention is a processof forming, on a base material, a cell culture patterning layer whereina cell adhesion portion having cell adhesive properties and a celladhesion-inhibiting portion having cell adhesion-inhibiting propertiescan be formed by action of a photocatalyst by energy irradiation,thereby forming a patterning substrate.

In the present process, the method for forming a cell culture patterninglayer on a base material to form a patterning substrate may be selectedfrom various methods in accordance with the kind of the cell culturepatterning layer. The method may be, for example, a wet process such asspin coating, spray coating, dip coating, roll coating, bead coating ordie coating, or a dry process such as PVD or CVD. The adsorption processalso can be preferably used.

This patterning substrate forming process can be classified into 6embodiments in accordance with the kind of the patterning substrate tobe formed.

(1) First Embodiment

First, the patterning substrate formed according to the first embodimentof the present process is a patterning substrate comprising a basematerial, and a cell culture patterning layer which is formed on thebase material and has a photocatalyst-containing cell adhesive layercomprising at least a photocatalyst and a cell adhesive material whichhas cell adhesive properties and is decomposed or denatured by action ofthe photocatalyst on the basis of energy irradiation.

The photocatalyst-containing cell adhesive layer contained in the cellculture patterning layer formed in the present embodiment comprisestherein the photocatalyst; therefore, in the energy irradiating processwhich will be detailed later, at the time of irradiating energy, thecell adhesive material in the layer can be decomposed or denatured byaction of the photocatalyst. It is therefore unnecessary to form a layercomprising any photocatalyst separately, or the like in the cell culturepatterning layer. Thus, the cell culture patterning layer can berendered a single layer. This is preferred from the viewpoint ofproduction efficiency and the like. If necessary, the cell culturepatterning layer may be formed to comprise a layer other than thephotocatalyst-containing cell adhesive layer.

The following will describe the base material used in the patterningsubstrate forming process of the present embodiment, and thephotocatalyst-containing cell adhesive layer formed in the presentembodiment.

(Base Material)

First, the base material used in the embodiment is described. The basematerial is not limited to any special kind if the cell culturepatterning layer having the photocatalyst-containing cell adhesive layerthat will be detailed later can be formed on this base material. Forexample, the following can be used: an inorganic material such as metal,glass, silicon; or an organic material such as plastic.

The flexibility and the like of the base material is appropriatelyselected in accordance with the kind or use purpose of the cell culturesubstrate to be finally obtained. The transparency of the base materialis appropriately selected in accordance with the kind of the cellculture substrate, the irradiation direction of energy irradiated todecompose or denature the cell adhesive material in the energyirradiating process, which will be detailed later, or other factors.When the energy is irradiated from the side of the base material, or thelike, the base material needs to be transparent.

In the present embodiment, the base material may be a flat base materialor a base material wherein one or more concave portions are formed. Thebase material may be a base material wherein a single concave portion isformed as shown in FIG. 7A, or a base material wherein plural concaveportions are formed as shown in FIG. 7B.

At this time, side walls of the base material having the concaveportion(s) may be treated in such a manner that thephotocatalyst-containing cell adhesive layer which will be detailedlater will not be formed thereon. Examples of the method for such atreatment include a method of using a mask or the like to cause amaterial having liquid repellency to adhere only onto the side walls byCVD; and a method of causing a material having liquid repellency toadhere onto the entire surface of the concave portion(s) and then usinga cylindrical mask or the like to conduct ultraviolet ray treatment,plasma treatment or some other treatment, thereby making only the bottomface(s) of the concave portion(s) lyophilic.

In the present embodiment, the base material may be washed with amedical liquid, such as an alkali solution, and may be subjected to drywashing, such as oxygen plasma treatment or ultraviolet treatment. Inthis case, the wettability of a coating solution for forming thephotocatalyst-containing cell adhesive layer is improved. Furthermore,this case has an advantage that the adhesive property of the basematerial to the photocatalyst-containing cell adhesive layer is improvedsince reactive functional groups are arranged on the surface of the basematerial.

On the base material according to the present embodiment, as needed, alight-shielding portion may be formed. The light-shielding portion thatcan be used in the embodiment, as far as it can shield energy that isirradiated on the patterning substrate, is not particularly restricted.For instance, a metal thin film that is made of chromium or the like andformed into a thickness of about 1000 to 2000 Å by a sputtering method,a vacuum deposition method or the like is formed and patterned to form ashielding portion. As the patterning method, an ordinary patterningmethod such as the sputtering can be used.

A method may be one by which a layer that contains light-shieldingparticles such as carbon particulates, metal oxides, inorganic pigmentsand organic pigments in a resin binder is formed in a pattern. As theresin binders that can be used, a polyimide resin, acrylic resin, epoxyresin, polyacrylamide, polyvinyl alcohol, gelatin, casein, cellulose andthe like can be used singularly or in combination of two or more kinds,and furthermore a photosensitive resin and an O/W emulsion type resincomposition such as emulsified reactive silicone can be used. Athickness of such resinous light-shielding portion can be set in therange of 0.5 to 10 μm. As a method of patterning such resinouslight-shielding portion, methods such as a photolithography method and aprinting method that are generally used can be used.

The light-shielding portions may be ones formed on the surface of theside at which the cell culture patterning layer which will be detailedlater is formed, or ones formed on the opposite surface.

In the case of forming the light-shielding portions, a primer layer maybe formed between the cell culture patterning layer and thelight-shielding portions. The effect and function of this primer layerare not necessarily clear, but would be as follows: the primer layerexhibits a function of preventing the diffusion of impurities from thelight-shielding portions and openings which are present between thelight-shielding portions, the impurities being factors for inhibitingthe decomposition or denaturation of the cell adhesive material in thephotocatalyst-containing cell adhesive layer by action of thephotocatalyst, in particular, residues generated when thelight-shielding portions are patterned, or metal, metal ion impurities,or the like. Accordingly, the formation of the primer layer makes itpossible to decompose or denature the cell adhesive material with highsensitivity in the energy irradiating process, which will detailedlater, so that a cell adhesion portion and a cell adhesion-inhibitingportion are formed in a highly precise pattern form.

The primer layer in the embodiment is a layer for preventing the effectof the photocatalyst from being affected by the impurities presentinside not only the light-shielding portions but also the openings madebetween the light-shielding portions. It is therefore preferred to formthe primer layer over the entire surface of the light-shielding portionsplus the openings.

The primer layer in the invention is not limited to any specialstructure if the primer layer is formed not to bring the light-shieldingportions and the cell culture patterning layer into contact with eachother.

A material that forms the primer layer, though not particularlyrestricted, is preferably an inorganic material that is not likely to bedecomposed by action of the photocatalyst. Specifically, amorphoussilica can be cited. When such amorphous silica is used, a precursor ofthe amorphous silica is preferably a silicon compound that isrepresented by a general formula, SiX₄, X being halogen, methoxy group,ethoxy group, acetyl group or the like, silanol that is a hydrolysatethereof, or polysiloxane having an average molecular weight of 3000 orless.

A film thickness of the primer layer is preferably in the range of 0.001to 1 μm and particularly preferably in the range of 0.001 to 0.1 μm.

It is preferred that on the base material a position-detecting mark isformed at a given location thereof. This makes the positioning of thebase material easy in the energy irradiating process or thecell-containing liquid applying process, which will be detailed later tomake it possible to form the cell adhesion-inhibiting portion at a givenlocation or applying the cell-containing liquid selectively and highlyprecisely only onto the cell adhesion portion. No special limitation isimposed on such position-detecting mark if the mark can be detected inthe energy irradiating process or the cell-containing liquid applyingprocess. Thus, the mark may be the same mark as is ordinarily used forthe positioning of substrates, or the like.

(Photocatalyst-Containing Cell Adhesive Layer)

The following will describe the photocatalyst-containing cell adhesivelayer formed in the present process. The photocatalyst-containing celladhesive layer is a layer comprising at least a photocatalyst and a celladhesive material which has cell adhesive properties and is decomposedor denatured by action of the photocatalyst on the basis of energyirradiation. In the embodiment, the method for forming the layer is notspecially limited if the method makes the formation of the layerpossible. For example, the layer can be formed by applying a coatingsolution for forming a photocatalyst-containing cell adhesive layerwhich comprises the photocatalyst and the cell adhesive material by theabove-mentioned applying method.

For example, when concave portions are made in the base material asdescribe above, it is possible to use: a casting process of droppingdown the coating solution for forming a photocatalyst-containing celladhesive layer into the concave portions, and drying the solution so asto form the photocatalyst-containing cell adhesive layer; an adsorbingprocess of dropping down the coating solution for forming aphotocatalyst-containing cell adhesive layer into the concave portionsof the base material, and washing the surface after a given time; orsome other process.

The film thickness of the photocatalyst-containing layer isappropriately selected in accordance with the kind of the cell culturesubstrate and other factors, and may be usually from about 0.01 to 1.0μm, preferably from about 0.1 to 0.3 μm.

The following will describe each of the materials used in thephotocatalyst-containing cell adhesive layer.

a. Cell Adhesive Material

First, the cell adhesive material used in the photocatalyst-containingcell adhesive layer formed in the embodiment is described. About thecell adhesive material, the kind thereof and others are not speciallylimited if the material has cell adhesive properties and is decomposedor denatured by action of a photocatalyst on the basis of energyirradiation. The wording “the material has cell adhesive properties”referred to herein means that the material adheres well to cells. When,for example, adhesive properties of the material to cells are differentin accordance with the kinds of the cells, the wording means that thematerial adheres well onto target one out of the cells.

The cell adhesive material used in the present embodiment has such celladhesive properties and can be decomposed or denatured by action of thephotocatalyst on the basis of energy irradiation such as to lose thecell adhesive properties or change to one that has the cell adhesioninhibiting properties that inhibit to adhere to cells.

As such materials having the cell adhesive properties, there are twokinds, one being materials having the cell adhesive properties owing tophysicochemical characteristics and the other being materials having thecell adhesive properties owing to biochemical characteristics.

As physicochemical factors that determine the cell adhesive propertiesof materials having the cell adhesive properties owing to thephysicochemical characteristics, the surface free energy, theelectrostatic interaction and the like can be cited. For instance, inthe case of the cell adhesive properties being determined by the surfacefree energy of the material, when the material has the surface freeenergy in a predetermined range, the adhesive properties between thecells and the material becomes good, and when it deviates from the aboverange the adhesive properties between the cells and material decreases.As such changes of the cell adhesive properties due to the surface freeenergy, experimental results such as shown in Data, for instance, CMCPublishing Co., Ltd. “Biomaterial no Saisentan”, Yoshito IKADA (editor),p. 109, lower part are known. As materials having the cell adhesiveproperties owing to such a factor, for instance, hydrophilicpolystyrene, poly (N-isopropyl acrylamide) and the like can be cited.When such a material is used, by action of the photocatalyst on thebasis of energy irradiation, for instance, a functional group on asurface of the material is substituted, decomposed or the like to causea change in the surface free energy, resulting in one that does not havethe cell adhesive properties or one that has the cell adhesioninhibiting properties.

When the adhesive properties between cells and a material is determinedowing to the electrostatic interaction or the like, for instance, thecell adhesive properties can be determined owing to an amount ofpositive electric charges and the like that the material has. Asmaterials having the cell adhesive properties owing to suchelectrostatic interaction, basic polymers such as polylysine, basiccompounds such as aminopropyltriethoxysilane,N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and condensates and thelike including these can be cited. When such materials are used, byaction of the photocatalyst on the basis of energy irradiation, theabove-mentioned materials are decomposed or denatured, thereby, forinstance, an amount of positive electric charges present on a surfacecan be altered, resulting in one that does not have the cell adhesiveproperties or one that has the cell adhesion inhibiting properties.

As materials having the cell adhesive properties owing to the biologicalcharacteristics, ones that are good in the adhesive properties withparticular cells, ones that are good in the adhesive properties withmany cells, or the like can be cited. Specifically, fibronectin,laminin, tenascin, vitronectin, RGD (arginine-glycine-asparagine acid)sequence containing peptide, YIGSR(tyrosine-isoleucine-glycine-serine-arginine) sequence containingpeptide, collagen, atelocollagen, gelatin, albumin, γ-globulin, and thelike can be cited. When such materials are used, by action of thephotocatalyst on the basis of energy irradiation, for instance, astructure of the material is partially destroyed, a principal chain isdestroyed or the like, resulting in one that does not have the celladhesive properties or one that has the cell adhesion inhibitingproperties.

The amount of the cell adhesive material is varied in accordance withthe kind of the material, or the like. Usually, the content bypercentage of the cell adhesive material in the photocatalyst-containingcell adhesive layer is preferably from 0.01 to 95% by weight, morepreferably from 1 to 10% by weight. This makes it possible to render theregion comprising the cell adhesive material a region good in celladhesive properties.

b. Photocatalyst

The following will describe the photocatalyst comprised in thephotocatalyst-containing cell adhesive layer formed in the presentembodiment. The photocatalyst is not limited to any special kind if thecell adhesive material can be decomposed or denatured by action of thisphotocatalyst on the basis of energy irradiation.

An action mechanism of the photocatalyst typical in titanium oxidedescribed below is not necessarily clear. However, it can be consideredthat a carrier generated by irradiation of light directly reacts with anearby compound or, owing to an active oxygen species generated underthe presence of oxygen, water, a chemical structure of an organicmaterial is caused to change. In the invention, the carrier isconsidered to affect on the cell adhesive material described above.

As the photocatalyst that can be used in the present embodiment,specifically, for instance, titanium dioxide (TiO₂), zinc oxide (ZnO),tin oxide (SnO₂) strontium titanate (SrTiO₃), tungsten oxide (WO₃),bismuth oxide (Bi₂O₃) and iron oxide (Fe₂O₃) that are known asphoto-semiconductors can be cited. These can be used singularly or incombination of at least two kinds.

In the present embodiment, in particular, titanium dioxide, owing to alarge band gap, chemical stability, non-toxicity, and easy availability,can be preferably used. There are two types of titanium dioxide, anatasetype and rutile type, and both can be used in the invention; however,the anatase type titanium dioxide is more preferable. An excitationwavelength of the anatase type titanium dioxide is 380 nm or less.

As such anatase type titanium dioxide, for instance, an anatase titaniasol of hydrochloric acid deflocculation type (trade name: STS-02,manufactured by ISHIHARA SANGYO KAISHA, LTD., average particle diameter:7 nm, and trade name: ST-KO1, manufactured by ISHIHARA SANGYO KAISHA,LTD.), an anatase titania sol of nitric acid deflocculation type(tradename: TA-15, manufactured by NISSAN CHEMICAL INDUSTRIES LTD.,average particle diameter: 12 nm) and the like can be cited.

The smaller is a particle diameter of the photocatalyst, the better,because a photocatalyst reaction is caused more effectively. An averageparticle diameter of the photocatalyst is preferably 50 nm or less, andone having an average particle diameter of 20 nm or less can beparticularly preferably used.

The content by percentage of the photocatalyst in thephotocatalyst-containing cell adhesive layer formed in the embodimentcan be set into a value from 5 to 95% by weight, preferably from 10 to60% by weight, more preferably from 20 to 40% by weight. This makes itpossible to decompose or denature the cell adhesive material inside theenergy-irradiated region in the photocatalyst-containing cell adhesivelayer in the energy irradiating process, which will be detailed later.

The photocatalyst used in the embodiment is preferably a photocatalystlow in cell adhesive properties. This makes it possible to use, as aregion low in cell adhesive properties, the region where the celladhesive material is decomposed or the like so that the photocatalyst isexposed.

c. Others

In the present embodiment, the photocatalyst-containing cell adhesivelayer may comprise, besides the cell adhesive material and thephotocatalyst, a binder for improving the strength or resistancethereof, or the like if necessary. In the embodiment, it is particularlypreferred to use, as the binder, a material having celladhesion-inhibiting properties for inhibiting adhesion onto cells atleast after the material is irradiated with energy. This makes itpossible to make low the cell adhesive properties of the regionirradiated with the energy in the energy irradiating process, which willbe detailed later. The material may be, for example, a material havingcell adhesion-inhibiting properties from before the material isirradiated with energy, or a material coming to have celladhesion-inhibiting properties by action of a photocatalyst on the basisof energy irradiation.

In the embodiment, the content by percentage of the binder in thephotocatalyst-containing cell adhesive layer is preferably from 5 to 95%by weight, more preferably from 40 to 90% by weight, even morepreferably from 60 to 80% by weight.

(2) Second Embodiment

The patterning substrate formed according to the second embodiment is apatterning substrate comprising a base material, and a cell culturepatterning layer which is formed on the base material and has aphotocatalyst-containing cell adhesion-inhibiting material layercomprising at least a photocatalyst and a cell adhesion-inhibitingmaterial which has cell adhesion-inhibiting properties of inhibitingadhesion onto cells and is decomposed or denatured by action of thephotocatalyst on the basis of energy irradiation.

The photocatalyst-containing cell adhesion-inhibiting material layerformed in the embodiment comprises therein the photocatalyst; therefore,in the energy irradiating process, which will be detailed later, at thetime of irradiating energy, the cell adhesion-inhibiting material in thelayer can be decomposed or denatured by action of the photocatalyst. Itis therefore unnecessary to form a layer comprising any photocatalystseparately in the cell culture patterning layer. Thus, the cell culturepatterning layer can be rendered a single layer. This is preferred fromthe viewpoint of production efficiency and the like. If necessary, thecell culture patterning layer may be formed to comprise a layer otherthan the photocatalyst-containing cell adhesion-inhibiting layer.

The following will describe the photocatalyst-containing celladhesion-inhibiting material layer formed in the present process. Thebase material used in the present process may be the same as used in thefirst embodiment. Thus, description thereof is omitted herein.

(Photocatalyst-Containing Cell Adhesion-Inhibiting Material Layer)

The photocatalyst-containing cell adhesion-inhibiting material layerformed in the process is a layer comprising at least a photocatalyst anda cell adhesion-inhibiting material which has cell adhesion-inhibitingproperties of inhibiting adhesion onto cells and is decomposed ordenatured by action of the photocatalyst on the basis of energyirradiation. In the embodiment, the method for forming the layer, or thelike is not specially limited if the method makes the formation of thelayer possible. For example, the layer can be formed by applying acoating solution for forming a photocatalyst-containing celladhesion-inhibiting material layer which comprises the photocatalyst andthe cell adhesion-inhibiting material by the above-mentioned applyingmethod, or the like.

For example, when concave portions are made in the base material asdescribe above, it is possible to use: a casting process of droppingdown the coating solution for forming a photocatalyst-containing celladhesion-inhibiting material layer into the concave portions, and dryingthe solution so as to form the photocatalyst-containing celladhesion-inhibiting material layer; an adsorbing process of droppingdown the coating solution for forming a photocatalyst-containing celladhesion-inhibiting material layer into the concave portions of the basematerial, and washing the surface after a given time; or some otherprocess.

The film thickness of the photocatalyst-containing layer isappropriately selected in accordance with the kind of the cell culturesubstrate and other factors, and may be usually from about 0.01 to 1.0μm, preferably from about 0.1 to 0.3 μm.

The following will describe each of the materials used in thephotocatalyst-containing cell adhesion-inhibiting layer formed in theembodiment. The photocatalyst used in the photocatalyst-containing celladhesion-inhibiting layer is the same as used in the first embodiment.Thus, description thereof is omitted herein.

a. Cell Adhesion-Inhibiting Material

First, the cell adhesion-inhibiting material used in thephotocatalyst-containing cell adhesion-inhibiting material layer formedin the embodiment is described.

About the cell adhesion-inhibiting material, the kind thereof and othersare not specially limited if the material has cell adhesion-inhibitingproperties of inhibiting adhesion onto cells and is decomposed ordenatured by action of a photocatalyst on the basis of energyirradiation.

Here, the wording “having the cell adhesion-inhibiting properties” meanshaving a property that inhibits cells from adhering to the celladhesion-inhibiting material, and, when cell adhesive properties aredifferent depending on the kind of cells and the like, means to have theproperty of inhibiting from adhering with a target cell.

As the cell adhesion-inhibiting material used in the present embodiment,one that has such cell adhesion-inhibiting properties and can bedecomposed or denatured by an action of the photocatalyst on the basisof energy irradiation to lose the cell adhesion-inhibiting properties orto become good in the cell adhesive properties can be used.

As such cell adhesion-inhibiting material, materials high in, forinstance, the hydration ability can be used. When the material high inthe hydration ability is used, water molecules gather in the vicinity ofthe cell adhesion-inhibiting material to form a hydrated layer.Normally, such material high in the hydration ability is higher in anadhesiveness with water molecules than that with cells; accordingly,cells cannot adhere to the material high in the hydration ability,resulting in one low in the cell adhesive properties. Here, “thehydration ability” means a property of hydrolyzing with water molecules,and “the hydration ability being high” means that it can readilyhydrolyze with water molecules.

As materials that are high in the hydration ability and can be used asthe cell adhesion-inhibiting material, for instance, polyethyleneglycol, amphoteric ion materials having a betaine structure or the like,and phosphatide-containing materials and the like can be cited. In thecase of such a material being used as the cell adhesion-inhibitingmaterial, when energy is irradiated in an energy irradiating process tobe described later, owing to an action of the photocatalyst, the celladhesion-inhibiting material is decomposed, denatured or the like, thehydrated layer on a surface comes off, and thereby the celladhesion-inhibiting material is rendered one that does not have the celladhesion-inhibiting properties.

In the present embodiment, as the cell adhesion-inhibiting material, asurfactant having a water repellent or oil repellent organic group thatcan be decomposed by an action of the photocatalyst also can be used. Assuch surfactant, hydrocarbons base surfactants such as the respectiveseries of NIKKO L, BL, BC, BO, and BB manufactured by Nikko ChemicalsCo., Ltd., and fluorine base or silicone base nonionic surfactants suchas ZONYL FSN and FSO manufacture by Du Pont Kabushiki Kaisya, SurflonS-141 and 145 manufactured by ASAHI GLASS CO., LTD., Megaface F-141 and144 manufactured by DAINIPPON INK AND CHEMICALS, Inc., FTERGENT F-200and F251 manufactured by NEOS, UNIDYNE DS-401 and 402 manufactured byDAIKIN INDUSTRIES, Ltd., and Fluorad FC-170 and 176 manufactured by 3Mcan be cited, and furthermore cationic surfactants, anionic surfactantsand amphoteric surfactants also can be used.

In the case of such a material being used as the celladhesion-inhibiting material to form the photocatalyst-containing celladhesion-inhibiting material layer, the cell adhesion-inhibitingmaterial is distributed unevenly on a surface. Thereby, the waterrepellency or oil repellency of the surface can be made higher;accordingly, one small in the interaction with cells and low in the celladhesive properties can be obtained. Furthermore, when energy isirradiated on the layer, by action of the photocatalyst, the celladhesion-inhibiting material is readily decomposed to expose thephotocatalyst, resulting in one that does not have the celladhesion-inhibiting properties.

In the present embodiment, as the cell adhesion-inhibiting material, onethat can be improved in the cell adhesive properties by action of thephotocatalyst on the basis of energy irradiation is particularlypreferably used. As such cell adhesion-inhibiting materials, forinstance, materials having the oil repellency or water repellency can becited.

In the case of, as the cell adhesion-inhibiting material, a materialhaving the water repellency or the oil repellency being used, owing tothe water repellency or the oil repellency of the celladhesion-inhibiting material, one in which an interaction such as ahydrophobic interaction between cells and the cell adhesion-inhibitingmaterial is small and the cell adhesive properties are low can beobtained.

As a material having such water repellency or the oil repellency, forinstance, one where a skeleton has such a high bond energy that cannotbe decomposed by action of the photocatalyst and that has a waterrepellent or oil repellent organic substitution group that can bedecomposed by action of the photocatalyst can be cited.

As one that has the skeleton having such high bond energy that cannot bedecomposed by action of the photocatalyst and the water repellent or oilrepellent organic substitution group that can be decomposed by action ofthe photocatalyst, for instance, (1) organopolysiloxane that exhibitslarge strength by hydrolyzing or polycondensating chloro, alkoxysilaneor the like owing to a sol-gel reaction and the like, and (2)organopolysiloxane or the like in which reactive silicones arecrosslinked can be cited.

In the case of the (1), it is preferable to be organopolysiloxane thatis a hydrolysis condensate or cohydrolysis condensate of at least onekind of silicon compounds expressed by a general formula:Y_(n)SiX_((4-n))(Here, Y denotes an alkyl group, fluoroalkyl group, vinyl group, aminogroup, phenyl group or epoxy group, or organic groups including these,and X denotes an alkoxyl group, acetyl group or halogen. n is an integerof 0 to 3). The number of carbons of the group expressed with Y ispreferably in the range of 1 to 20, and the alkoxy group expressed withX is preferably a methoxy group, ethoxy group, propoxy group or butoxygroup.

As the organic groups, in particular, polysiloxane containing afluoroalkyl group can be preferably used. Specifically, a hydrolysiscondensate or cohydrolysis condensate of at least one kind offluoroalkylsilanes below can be cited. Ones generally known as thefluorinated silane coupling agents can be used.

CF₃(CF₂)₃CH₂CH₂Si(OCH₃)₃;

CF₃(CF₂)₅CH₂CH₂Si(OCH₃)₃;

CF₃(CF₂)₇CH₂CH₂Si(OCH₃)₃;

CF₃(CF₂)₉CH₂CH₂Si(OCH₃)₃;

(CF₃)₂CF(CF₂)₄CH₂CH₂Si(OCH₃)₃;

(CF₃)₂CF(CF₂)₆CH₂CH₂Si(OCH₃)₃;

(CF₃)₂CF(CF₂)₈CH₂CH₂Si(OCH₃)₃;

CF₃(C₆H₄)C₂H₄Si(OCH₃)₃;

CF₃(CF₂)₃(C₆H₄)C₂H₄Si(OCH₃)₃;

CF₃(CF₂)₅(C₆H₄)C₂H₄Si(OCH₃)₃;

CF₃(CF₂)₇(C₆H₄)C₂H₄Si(OCH₃)₃;

CF₃(CF₂)₃CH₂CH₂SiCH₃(OCH₃)₂;

CF₃(CF₂)₅CH₂CH₂SiCH₃(OCH₃)₂;

CF₃(CF₂)₇CH₂CH₂SiCH₃(OCH₃)₂;

CF₃(CF₂)₉CH₂CH₂SiCH₃(OCH₃)₂;

(CF₃)₂CF(CF₂)₄CH₂CH₂SiCH₃(OCH₃)₂;

(CF₃)₂CF(CF₂)₆CH₂CH₂SiCH₃(OCH₃)₂;

(CF₃)₂CF(CF₂)₈CH₂CH₂SiCH₃(OCH₃)₂;

CF₃(C₆H₄)C₂H₄SiCH₃(OCH₃)₂;

CF₃(CF₂)₃(C₆H₄)C₂H₄SiCH₃(OCH₃)₂;

CF₃(CF₂)₅(C₆H₄)C₂H₄SiCH₃(OCH₃)₂;

CF₃(CF₂)₇(C₆H₄)C₂H₄SiCH₃(OCH₃)₂;

CF₃(CF₂)₃CH₂CH₂Si(OCH₂CH₃)₃;

CF₃(CF₂)₅CH₂CH₂Si(OCH₂CH₃)₃;

CF₃(CF₂)₇CH₂CH₂Si(OCH₂CH₃)₃;

CF₃(CF₂)₉CH₂CH₂Si(OCH₂CH₃)₃;

CF₃(CF₂)₇SO₂N(C₂H₅) C₂H₄CH₂Si(OCH₃)₃

When polysiloxane containing such a fluoroalkyl group is used as thecell adhesion-inhibiting material, one high in the water repellency oroil repellency can be formed, resulting in rendering one small in theinteraction with cells and low in the cell adhesive properties.Furthermore, when energy is irradiated on such a material, readily,fluorine and the like can be removed and an OH group and the like can beintroduced on a surface to render the interaction with cells larger;accordingly, the cell adhesive properties can be made good.

As the reactive silicone according to the (2), compounds having askeleton expressed by a general formula below can be cited.

In the above general formula, n denotes an integer of 2 or more, R¹ andR² each represents a substituted or nonsubstituted alkyl group, alkenylgroup, aryl group or cyanoalkyl group having 1 to 20 carbons, and avinyl, phenyl and halogenated phenyl occupy 40% or less by mole ratio toa total mole. Furthermore, one in which R¹ and R² each is a methyl groupis preferable because the surface energy is the lowest, and a methylgroup is preferably contained 60% or more by mole ratio. Stillfurthermore, a chain terminal or side chain has at least one or morereactive group such as a hydroxyl group in a molecular chain.

Together with the organopolysiloxane, a stable organosilicium compoundthat does not cause a crosslinking reaction such as dimethylpolysiloxanemay be blended separately.

When such reactive silicone is used, one high in the water repellency oroil repellency can be obtained; that is, one small in the interactionwith cells and low in the cell adhesive properties can be obtained.Furthermore, when energy is irradiated on such a material, readily, asubstituent group can be removed and an OH group and the like can beintroduced on a surface to render the interaction with cells larger;accordingly, the cell adhesive properties can be made good.

In the case of using as the cell adhesion-inhibiting material apolysiloxane or reactive silicone as described above, the regionirradiated with energy in the energy irradiating process can be rendereda region having hydrophilicity, and the cell adhesion-inhibiting portionnot irradiated with the energy can be rendered a region having waterrepellency. This gives an advantage that when a cell-containing liquidis applied by the region-selecting applying method in thecell-containing liquid applying process, which will be detailed later,cells can be caused to adhere in a more highly precise pattern formsince the cells do not adhere onto the cell adhesion-inhibiting portionbut the cells adhere only onto the cell adhesion portion.

The above-mentioned word “hydrophilicity” means a small contact anglewith water, and the wordings “water repellency” means a large contactangle with water. Specifically, in the region not irradiated withenergy, that is, the cell adhesion-inhibiting portion having waterrepellency, the contact angle with water is preferably 100° or more,more preferably from 110 to 130°. If the contact angle with water issmaller than this range, the cell-containing liquid applied by theregion-selecting applying method in the cell-containing liquid applyingprocess, which will be detailed later, may adhere onto the celladhesion-inhibiting portion. The water repellency and oil repellency ofthe surface can be made higher by, for example, a method described in“Ogawa et al., Japanese Journal of Applied Physics P2, 32, (1993)L614-”, for instance, a method of subjecting a base material toreduced-pressure plasma treatment to make fine irregularities in thebase material, and further applying a water repellent and oil repellentmaterial onto the base material. In the case of using such a surface,the contact angle of the cell adhesion-inhibiting portion with water canbe made as high as about 160°.

In the region irradiated with energy, that is, the cell adhesion portionhaving hydrophilicity, the contact angle with water is preferably 50° orlower, more preferably from 10 to 40°, even more preferably from 15 to30°. If the contact angle with water is larger than the range, the celladhesion portion repels the cell-containing liquid applied by theregion-selecting applying method in the cell-containing liquid applyingprocess, which will be detailed later, so that cells may not be causedto adhere onto the portion.

The contact angle with water referred to herein is a result obtained byusing a contact angle measuring device (CA-Z model, manufactured byKyowa Interface Science Co., Ltd.) to measure the contact angle of thematerial with water or a liquid having a contact angle equivalent tothat of water (after 30 seconds from the time when droplets of theliquid are dropped down from its micro syringe), or a value obtainedfrom a graph prepared from the result.

The content by percentage of the cell adhesion-inhibiting material inthe photocatalyst-containing cell adhesion-inhibiting material layer ispreferably from 0.01 to 95% by weight, more preferably from 1 to 10% byweight. This makes it possible to render the region comprising the celladhesion-inhibiting material a region low in cell adhesive properties.

The cell adhesion-inhibiting material preferably has surface activity.This makes it possible that, for example, when a coating solution, forforming the photocatalyst-containing cell adhesion-inhibiting materiallayer, which comprises the cell adhesion-inhibiting material is applied,then dried and the like, the ratio of the material distributed unevenlyin the surface of the coating film becomes high so that good celladhesion-inhibiting properties can be obtained.

(Others)

The photocatalyst-containing cell adhesion-inhibiting material layer inthe present embodiment may comprise such as a binder in accordance withproperties required for a coating liquid for patterning substrate, forexample, the coating property for forming this layer, and the strengthor resistance of the layer. The cell adhesion-inhibiting material mayfulfill a part of the binder.

The binder may be, for example, a compound having a high bonding energysuch that the main skeleton thereof is not decomposed by action of theabove-mentioned photocatalyst. A specific example thereof is apolysiloxane having no organic substituent or having one or more organicsubstituents to such a degree that no effect is produced on the celladhesive property. The polysiloxane can be obtained by hydrolyzing orpolycondensing tetramethoxysilane, tetraethoxysilane or the like.

In the embodiment, the content by percentage of the binder in thephotocatalyst-containing cell adhesion-inhibiting material layer ispreferably from 5 to 95% by weight, more preferably from 40 to 90% byweight, even more preferably from 60 to 80% by weight. This makes itpossible to exhibit various properties.

In the embodiment, it is particularly preferred that thephotocatalyst-containing cell adhesion-inhibiting material layercomprises a cell adhesive material having cell adhesive properties atleast after the material is irradiated with energy. This makes itpossible that the photocatalyst-containing cell adhesion-inhibitingmaterial layer makes the cell adhesive properties of the regionirradiated with the energy better in the energy irradiating process,which will be detailed later. The cell adhesive material may be amaterial used as the above-mentioned binder, or a material usedindependently of the binder. The cell adhesive material may be, forexample, a material having good cell adhesive properties even before thematerial is irradiated with energy, or a material coming to have goodcell adhesive properties by action of a photocatalyst on the basis ofenergy irradiation. The wording “a material having cell adhesiveproperties” referred to herein means that the material adhere well ontocells. When adhesive properties of the material to cells are differentin accordance with the kinds of the cells or the like, the wording meansthat the material adheres well onto target one out of the cells.

In the present embodiment, as far as the cell adhesive material has goodcell adhesive properties at least after energy is irradiated, it may beone of which cell adhesive properties are made good owing to physicalinteractions such as an hydrophobic interaction, electrostaticinteraction, hydrogen bonding, van der Waals force and the like or owingto biological characteristics.

In the embodiment, the content by percentage of the cell adhesivematerial in the photocatalyst-containing cell adhesion-inhibitingmaterial layer is preferably from 0.01 to 95% by weight, more preferablyfrom 1 to 10% by weight. This makes it possible that thephotocatalyst-containing cell adhesion-inhibiting material layer makesthe cell adhesive properties of the region irradiated with energy betterin the energy irradiating process, which will be detailed later. In thecase of using, as the cell adhesive material, a material having goodcell adhesive properties even before the material is irradiated withenergy, it is preferred that the cell adhesive material is contained tosuch a degree that the cell adhesion-inhibiting properties of the celladhesion-inhibiting material is not inhibited in the region notirradiated with energy.

(3) Third Embodiment

The patterning substrate obtained according to the third embodiment ofthe present process is a patterning substrate comprising a basematerial, and a cell culture patterning layer which is formed on thebase material and has a photocatalyst-containing layer comprising atleast a photocatalyst and a cell adhesive layer formed on thephotocatalyst-containing layer, wherein the cell adhesive layercomprises at least a cell adhesive material which has cell adhesiveproperties and is decomposed or denatured by action of the photocatalyston the basis of energy irradiation.

In the embodiment, the cell culture patterning layer has the celladhesive layer and the photocatalyst-containing layer; therefore, in theenergy irradiating process, which will be detailed later, at the time ofirradiating energy, the cell adhesive material in the cell adhesivelayer can be decomposed or denatured by action of the photocatalystcomprised in the photocatalyst-containing layer, so that a celladhesion-inhibiting portion can be formed.

According to the present embodiment, the cell adhesive layer is formedon the photocatalyst-containing layer; therefore, at the time ofculturing cells, the cells do not contact the photocatalyst directly,thereby producing an advantage of making low the possibility that thecells are affected by the photocatalyst with the passage of time.

The following will describe the cell adhesive layer and thephotocatalyst-containing layer in the cell culture patterning layerformed according to the embodiment. The base material used in theembodiment may be the same as described in the first embodiment. Thus,description thereof is omitted herein.

(Cell Adhesive Layer)

First, the cell adhesive layer comprised in the cell culture patterninglayer formed in the embodiment is described. The cell adhesive layer isa layer formed on the photocatalyst-containing layer which will bedetailed later, and is a layer comprising at least a cell adhesivematerial which has cell adhesive properties and is decomposed ordenatured by action of a photocatalyst on the basis of energyirradiation.

In the embodiment, the method for forming the layer or the like is notspecially limited if the method makes the formation of the layerpossible. For example, the layer can be formed by applying a coatingsolution for forming a cell adhesive layer, which comprises the celladhesive material by the above-mentioned applying method, or othermethod.

When concave portions are made in the base material as describe above,it is possible to use: a casting process of forming aphotocatalyst-containing layer, which will be detailed later, onto theconcave portions in the base material, dropping down the coatingsolution for forming a cell adhesive layer, into the concave portions,and drying the solution so as to form the cell adhesive layer; anadsorbing process of forming a photocatalyst-containing layer, whichwill be detailed later, onto the concave portions in the base material,dropping down the coating solution for forming a cell adhesive layer,into the concave portions, and washing the surface after a given time;or some other process.

The film thickness of such cell adhesive layer is appropriately selectedin accordance with the kind of the cell culture substrate and otherfactors, and may be usually from about 0.001 to 1.0 μm, preferably fromabout 0.005 to 0.1 μm.

Specific examples of the cell adhesive material used in the celladhesive layer formed in the embodiment may be those equivalent to thoseof the cell adhesive material used in the photocatalyst-containing celladhesive layer in the first embodiment. Thus, detailed descriptionthereof is omitted herein. It is preferred that the cell adhesive layerin the embodiment also comprises a material having the celladhesion-inhibiting properties described about thephotocatalyst-containing cell adhesive layer in the first embodiment.This makes it possible to make the cell adhesive properties of theregion irradiated with energy low in the energy irradiating process.

(Photocatalyst-Containing Layer)

The following will describe the photocatalyst-containing layer comprisedin the cell culture patterning layer formed in the embodiment. Thephotocatalyst-containing layer is not limited to any special kind if thelayer is a layer comprising a photocatalyst. The layer may be a layermade only of the photocatalyst, or a layer comprising one or moredifferent components, such as a binder, also.

The photocatalyst used in the embodiment may be the same as used in thephotocatalyst-containing cell adhesive layer in the first embodiment. Inthe embodiment also, titanium oxide is particularly preferred.

It is advantageous from the viewpoint of costs to use thephotocatalyst-containing layer made only of a photocatalyst since theefficiency of decomposing or denaturing the cell adhesive material inthe cell adhesive layer is improved to make the time for the treatmentshorter or the like. On the other hand, the use of thephotocatalyst-containing layer made of a photocatalyst and a bindergives an advantage of making the formation of thephotocatalyst-containing layer easy.

An example of the method for forming the photocatalyst-containing layermade only of a photocatalyst may be a vacuum film-forming method such assputtering, CVD or vacuum vapor deposition. The formation of thephotocatalyst-containing layer by the vacuum film-forming method makesit possible to render the layer a homogeneous photocatalyst-containinglayer made only of a photocatalyst, thereby decomposing or denaturingthe cell adhesive material homogeneously, and further thereby making thedecomposition or denaturation of the cell adhesive material moreeffective in this case than in the case of using a binder also since thelayer is made only of the photocatalyst. When concave portions areformed on the base material mentioned above, thephotocatalyst-containing layer can be formed by the above-mentionedmethod.

Another example of the method for forming the photocatalyst-containinglayer made only of a photocatalyst, is the following method: in the casethat the photocatalyst is, for example, titanium dioxide, amorphoustitania is formed on the base material and next fired so as tophase-change the titania to crystalline titania. The amorphous titaniaused in this case can be obtained, for example, by hydrolysis ordehydration condensation of an inorganic salt of titanium, such astitanium tetrachloride or titanium sulfate, or hydrolysis or dehydrationcondensation of an organic titanium compound, such astetraethoxytitanium, tetraisopropoxytitanium, tetra-n-propoxytitanium,tetrabutoxytitanium or tetramethoxytitanium, in the presence of an acid.Next, the resultant is fired at 400 to 500° C. so as to be denatured toanatase type titania, and fired at 600 to 700° C. so as to be denaturedto rutile type titania.

In the case of using a binder together with the above-mentionedphotocatalyst, the binder is preferably a binder having a high bondingenergy, wherein its main skeleton is not decomposed by photoexcitationof the photocatalyst. Examples of such a binder include theorganopolysiloxanes described in the above-mentioned item “Cell AdhesiveLayer”.

In the case of using such an organopolysiloxane as the binder, thephotocatalyst-containing layer can be formed by dispersing aphotocatalyst, the organopolysiloxane as the binder, and optionaladditives if needed into a solvent to prepare a coating liquid, andapplying this coating liquid onto the base material. The used solvent ispreferably an alcoholic based organic solvent such as ethanol orisopropanol. The application can be performed by a known coating methodsuch as spin coating, spray coating, dip coating, roll coating, or beadcoating. When the coating liquid contains an ultraviolet curablecomponent as the binder, the photocatalyst-containing layer can beformed by curing the coating liquid through the irradiation ofultraviolet rays onto the liquid.

When concave portions are formed on the base material mentioned above,it is allowable to perform such as a casting method of dropping down theabove-mentioned coating liquid into the concave portions and then dryingthe liquid to form the photocatalyst-containing layer.

As the binder, an amorphous silica precursor can be used. This amorphoussilica precursor is preferably a silicon compound represented by thegeneral formula SiX₄, wherein X are a halogen, a methoxy group, anethoxy group, an acetyl group or the like; a silanol which is ahydrolyzate thereof; or a polysiloxane having an average molecularweight of 3000 or less.

Specific examples thereof include such as tetraethoxysilane,tetraisopropoxysilane, tetra-n-propoxysilane, tetrabutoxysilane, andtetramethoxysilane. In this case, the photocatalyst-containing layer canbe formed by dispersing the amorphous silica precursor and particles ofa photocatalyst homogeneously into a non-aqueous solvent, hydrolyzingthe precursor with water content in the air to form a silanol onto atransparent base material, and then subjecting the silanol todehydration polycondensation at room temperature. When the dehydrationpolycondensation of the silanol is performed at 100° C. or higher, thepolymerization degree of the silanol increases so that the strength ofthe film surface can be improved. A single kind or two or more kinds ofthis binding agent may be used.

The content by percentage of the photocatalyst in thephotocatalyst-containing layer is from 5 to 60% by weight, preferablyfrom 20 to 40% by weight. The thickness of the photocatalyst-containinglayer is preferably from 0.05 to 10 μm.

Besides the above-mentioned photocatalyst and binder, the surfactant andso on described in the above-mentioned item “Cell Adhesive Layer” can beincorporated into the photocatalyst-containing layer.

In the present embodiment, one or more light-shielding portions may beformed on the photocatalyst-containing layer, as described above.According to this, in the energy irradiating process, which will bedetailed later, when the entire surface of the cell adhesive layer isirradiated with energy, the cell adhesive material contained in regionsof the cell adhesive layer other than the regions thereof on which thelight-shielding portions are formed can be decomposed or denaturedwithout exciting the photocatalyst in the regions on which thelight-shielding portions are formed. This case has an advantage that thedirection in which the energy is irradiated is not particularly limitedsince the photocatalyst in the regions where light-shielding portionsare formed is not excited.

Such light-shielding portions may be the same as described in the item“Base Material” in the first embodiment. Thus, detailed descriptionthereof is omitted herein.

(4) Fourth Embodiment

The patterning substrate formed in the fourth embodiment of the presentprocess is a patterning substrate comprising a base material, and a cellculture patterning layer which is formed on the base material and has aphotocatalyst-containing layer comprising at least a photocatalyst and acell adhesion-inhibiting material layer formed on thephotocatalyst-containing layer, wherein the cell adhesion-inhibitingmaterial layer comprises a cell adhesion-inhibiting material which hascell adhesion-inhibiting properties of inhibiting adhesion onto thecells and is decomposed or denatured by action of the photocatalyst onthe basis of energy irradiation.

In the embodiment, the cell culture patterning layer has the celladhesion-inhibiting material layer and the photocatalyst-containinglayer; therefore, in the energy irradiating process, which will bedetailed later, at the time of irradiating energy, the celladhesion-inhibiting material in the cell adhesion-inhibiting materiallayer can be decomposed or denatured by action of the photocatalystcomprised in the photocatalyst-containing layer, so that a cell adhesionportion can be formed.

The following will describe the cell adhesion-inhibiting material layerin the cell culture patterning layer formed in the embodiment. The basematerial used in the embodiment may be the same as described in thefirst embodiment. The photocatalyst-containing layer may be the same asdescribed in the third embodiment. Thus, description thereof is omittedherein.

(Cell Adhesion-Inhibiting Material Layer)

The cell adhesion-inhibiting material layer formed in the embodiment isa layer formed on the photocatalyst-containing layer, and is not limitedto any special kind if the layer is a layer comprising a celladhesion-inhibiting material which has cell adhesion-inhibitingproperties of inhibiting adhesion onto cells and is decomposed ordenatured by action of a photocatalyst on the basis of energyirradiation.

In the embodiment, the method for forming the layer is not speciallylimited if the method makes the formation of the layer possible. Forexample, the layer can be formed by applying a coating solution forforming a cell adhesion-inhibiting material layer, which comprises thecell adhesion-inhibiting material by the above-mentioned applyingmethod, or other method.

When concave portions are made in the base material as describe above,it is possible to use: a casting process of forming aphotocatalyst-containing layer which will be detailed later onto theconcave portions in the base material, dropping down the coatingsolution for forming a cell adhesion-inhibiting material layer, into theconcave portions, and drying so as to form the cell adhesion-inhibitingmaterial layer; an adsorbing process of forming aphotocatalyst-containing layer which will be detailed later onto theconcave portions in the base material, dropping down the coatingsolution for forming a cell adhesion-inhibiting material layer, andwashing the surface after a given time; or some other process.

The film thickness of the cell adhesion-inhibiting material layer isappropriately selected in accordance with the kind of the cell culturesubstrate and other factors, and may be usually from about 0.01 to 1.0μm, preferably from about 0.1 to 0.3 μm.

Specific examples of the cell adhesion-inhibiting material used in thecell adhesion-inhibiting material layer formed in the embodiment may beequivalent to those of the cell adhesion-inhibiting material used in thephotocatalyst-containing cell adhesion-inhibiting material layer in thesecond embodiment. Thus, detailed description thereof is omitted herein.It is preferred that the cell adhesion-inhibiting material layer in theembodiment also comprises the cell adhesive material described about thephotocatalyst-containing cell adhesion-inhibiting material layer in thesecond embodiment. This makes it possible to make the cell adhesiveproperties of the region irradiated with energy high in the energyirradiating process, which will be detailed later.

In the embodiment also, it is preferred to use, as the celladhesion-inhibiting material, a material capable of rendering the regionirradiated with energy a hydrophilic region and making the celladhesion-inhibiting portion not irradiated with the energy waterrepellent in the energy irradiating process which will be detailedlater. According to this, at time of applying a cell-containing liquidby a region-selecting applying method in the cell-containing liquidapplying process, which will be detailed later, cells do not adhere ontothe cell adhesion-inhibiting portion and cells adhere only onto the celladhesion portion, thereby giving an advantage that the cells can beadhered in a more highly precise pattern form.

Specifically, in the region not irradiated with energy, that is, in thecell adhesion-inhibiting portion having water repellency, the contactangle with water is preferably 100° or more, more preferably from 110 to130°. If the contact angle with water is smaller than this range, thecell-containing liquid applied by the region-selecting applying methodin the cell-containing liquid applying process, which will be detailedlater, may adhere onto the cell adhesion-inhibiting portion. In the caseof forming irregularities in the base material and measuring the contactangle thereof with water, the contact angle can be made as high as about160°, as described above.

In the region irradiated with energy, that is, the cell adhesion portionhaving hydrophilicity, the contact angle with water is preferably 50° orlower, more preferably from 10 to 40°, even more preferably from 15 to30°. If the contact angle with water is larger than the range, the celladhesion portion repels the cell-containing liquid applied by theregion-selecting applying method in the cell-containing liquid applyingprocess, which will be detailed later, so that cells may not be causedto adhere onto the portion. The contact angle with water referred toherein can be measured by the above-mentioned method.

(5) Fifth Embodiment

The patterning substrate formed according to the fifth embodiment of thepresent process is a patterning substrate comprising a base material anda cell culture patterning layer which is formed on the base material andhas a cell adhesive layer comprising a cell adhesive material that hascell adhesive properties and is decomposed or denatured by action of aphotocatalyst on the basis of energy irradiation.

In the embodiment, the cell adhesive layer comprises therein the celladhesive material, which is decomposed or denatured by action of aphotocatalyst on the basis of energy irradiation; therefore, the celladhesive material in the cell adhesive layer can be decomposed ordenatured by energy irradiation by use of a photocatalyst-containinglayer side substrate having a base body and a photocatalyst-containinglayer comprising the photocatalyst in the energy irradiating process,which will be detailed later. Consequently, a non-cell adhesion portioncan be formed. In the embodiment, the cell culture patterning layer doesnot need to comprise therein any photocatalyst; therefore, adeterioration of the layer with the passage of time can be made small.

The base material used in the embodiment may be the same as described inthe first embodiment. The cell adhesive layer may be the same asdescribed in the third embodiment. Thus, detailed description thereof isomitted herein.

(6) Sixth Embodiment

The patterning substrate obtained according to the sixth embodiment ofthe present process is a patterning substrate comprising a base materialand a cell culture patterning layer which is formed on the base materialand has a cell adhesion-inhibiting material layer comprising a celladhesion-inhibiting material that has cell adhesion-inhibitingproperties of inhibiting adhesion onto the cells and is decomposed ordenatured by action of a photocatalyst on the basis of energyirradiation.

In the embodiment, the cell adhesion-inhibiting material layer comprisestherein the cell adhesion-inhibiting material, which is decomposed ordenatured by action of a photocatalyst on the basis of energyirradiation; therefore, the cell adhesion-inhibiting material in thecell adhesion-inhibiting material layer can be decomposed or denaturedby energy irradiation by use of a photocatalyst-containing layer sidesubstrate having a base body and a photocatalyst-containing layercomprising the photocatalyst in the energy irradiating process, whichwill be detailed later. In the embodiment, the cell culture patterninglayer does not need to comprise therein any photocatalyst; therefore, adeterioration of the layer with the passage of time can be made small.

The base material used in the embodiment may be the same as described inthe first embodiment. The cell adhesion-inhibiting material layer may bethe same as described in the fourth embodiment. Thus, detaileddescription thereof is omitted herein.

In the embodiment also, it is preferred to use, as the celladhesion-inhibiting material, a material capable of rendering the regionirradiated with energy a hydrophilic region and making the celladhesion-inhibiting portion not irradiated with the energy waterrepellent in the energy irradiating process. According to this, at timeof applying a cell-containing liquid by a region-selecting applyingmethod in the cell-containing liquid applying process, which will bedetailed later, cells do not adhere onto the cell adhesion-inhibitingportion and cells adhere only onto the cell adhesion portion, therebygiving an advantage that the cells can be adhered in a more highlyprecise pattern form.

Specifically, in the region not irradiated with energy, that is, in thecell adhesion-inhibiting portion having water repellency, the contactangle with water is preferably 100° or more, more preferably from 110 to130°. If the contact angle with water is smaller than this range, thecell-containing liquid applied by the region-selecting applying methodin the cell-containing liquid applying process, which will be describedlater, may adhere onto the cell adhesion-inhibiting portion. In the caseof forming irregularities in the base material and measuring the contactangle thereof with water, the contact angle can be made as high as about160°, as described above.

In the region irradiated with energy, that is, the cell adhesion portionhaving hydrophilicity, the contact angle with water is preferably 50° orlower, more preferably from 10 to 40°, even more preferably from 15 to30°. If the contact angle with water is larger than the range, the celladhesion portion repels the cell-containing liquid applied by theregion-selecting applying method in the cell-containing liquid applyingprocess, which will be described later, so that cells may not be causedto adhere onto the portion. The contact angle with water referred toherein can be measured by the above-mentioned method.

2. Energy Irradiating Process

The following will describe the energy irradiating process in the methodof the invention for producing a cell culture substrate. This energyirradiating process is a process of irradiating energy onto the cellculture patterning layer, thereby forming the cell adhesion portion andthe cell adhesion-inhibiting portion in a pattern form by action of aphotocatalyst.

The present process can be similarly classified into six embodiments,for irradiating energy and forming the cell adhesion portion and thecell adhesion-inhibiting portion, in accordance with the six embodimentsof the patterning substrate.

(1) First Embodiment

The first embodiment of the present process is performed when thepatterning substrate is the first embodiment, and is a process ofirradiating energy from a given direction, thereby forming a patterncomposed of a cell adhesion-inhibiting portion where the cell adhesivematerial comprised in the photocatalyst-containing cell adhesive layeris decomposed or denatured, and a cell adhesion portion which is otherthan the cell adhesion-inhibiting portion.

As shown in, for example, FIGS. 1A and 1B, the energy irradiatingprocess of the embodiment is a process of irradiating energy 4 onto aphotocatalyst-containing cell adhesive layer 2 which is formed on a basematerial 1 and comprises the cell adhesive material and a photocatalystin a pattern form for forming cell adhesion-inhibiting portions throughsuch as a photomask 3 (FIG. 1A). This makes it possible to form celladhesion-inhibiting portions 5 where the cell adhesive material in theregion irradiated with the energy is decomposed or denatured and celladhesion portions 6 where the cell adhesive material remains withoutbeing irradiated with the energy (FIG. 1B).

In the embodiment, energy is irradiated in a pattern form for formingthe cell adhesion-inhibiting portion, thereby exciting the photocatalystcomprised in the photocatalyst-containing cell adhesive layer so thatthe cell adhesive material can be decomposed or denatured. It istherefore possible to render the region irradiated with the energy thecell adhesion-inhibiting portion, where the cell adhesive material isdecomposed or denatured, and render the region not irradiated with theenergy the cell adhesion portion, where the cell adhesive materialremains. At this time, the cell adhesion-inhibiting portion contains thephotocatalyst, a decomposition or denaturation product of the celladhesive material, and so on.

The energy irradiation (exposure) mentioned in the present embodiment isa concept that includes all energy line irradiation that can decomposeor denature the cell adhesive material by action of a photocatalyst onthe basis of energy irradiation, and is not restricted to lightirradiation.

Normally, a wavelength of light used in such energy irradiation is setin the range of 400 nm or less, and preferably in the range of 380 nm orless. This is because, as mentioned above, the photocatalyst that ispreferably used as a photocatalyst is titanium dioxide, and as energythat activates a photocatalyst action by the titanium oxide, lighthaving the above-mentioned wavelength is preferable.

As a light source that can be used in such energy irradiation, a mercurylamp, metal halide lamp, xenon lamp, excimer lamp and other variouskinds of light sources can be cited.

The method for the exposure may be a method of using a laser, such as anexcimer laser or YAG laser, to draw and irradiate energy in a patternform, besides a method of using a light source as described above todraw and irradiate energy in a pattern form through a photomask. Whenthe base material has light-shielding portions in the same pattern formas the cell adhesion portions have, as described above, the exposure canbe performed by irradiating the entire surface of the cell culturepatterning substrate from the base material side thereof. These caseshave an advantage that a photomask, and positioning and other steps areunnecessary.

When the base material has one or more concave portions and thephotocatalyst-containing cell adhesive layer is formed in the concaveportion(s), as described above, the exposure may be performed by any oneof the above-mentioned methods. For instance, in the case of the pluralconcave portions, for example, the exposure may be performed into theform of patterns different from each other for the individual concaveportions. Examples of the method for performing the exposure into theform of patterns different from each other for the individual concaveportions as described above include a method of arranging differentmasks for the individual concave portions to irradiate energy; and amethod of arranging a chromium mask, a stencil mask or the like at thetip of an optical fiber to irradiate energy.

In order not to irradiate any energy onto side walls of the concaveportions, the method for the exposure may be, for example, a method ofusing a cylindrical mask to irradiate energy only onto the bottom facesof the concave portions.

The irradiation quantity of the energy at the time of theenergy-irradiation is set to a value necessary for decomposing ordenaturing the cell adhesive material by action of the photocatalyst.

It is preferred to irradiate the energy while heating thephotocatalyst-containing cell adhesive layer, which comprises aphotocatalyst, at this time since the patterning sensitivity can beraised and the cell adhesive material can be effectively decomposed ordenatured. Specifically, it is preferred to heat the layer at atemperature of 30 to 80° C.

The energy irradiation through the photomask in the embodiment may beirradiated from either of the side of the base material and the side ofthe photocatalyst-containing cell adhesive layer when the base materialis transparent. On the other hand, when the base material is opaque, theenergy needs to be irradiated from the side of thephotocatalyst-containing cell adhesive layer.

In the embodiment, it is allowable to cut a part of the patterningsubstrate where the cell adhesion portion and the celladhesion-inhibiting portion are formed in a pattern form, which may bereferred to as the cell culture patterning substrate hereinafter, stickthis part of the cell culture patterning substrate onto such as thebottom of a concave-form base material, and subsequently perform thecell-containing liquid applying process, which will be detailed later.

(2) Second Embodiment

The second embodiment of the present process is performed when thepatterning substrate is the second embodiment, and is a process ofirradiating energy from a given direction, thereby forming a patterncomposed of a cell adhesion portion where the cell adhesion-inhibitingmaterial comprised in the photocatalyst-containing celladhesion-inhibiting material layer is decomposed or denatured, and acell adhesion-inhibiting portion which is other than the cell adhesionportion.

In the embodiment, energy is irradiated in a pattern form for formingthe cell adhesion portion, thereby exciting the photocatalyst comprisedin the photocatalyst-containing cell adhesion-inhibiting material layerso that the cell adhesion-inhibiting material can be decomposed ordenatured. It is therefore possible to render the region irradiated withthe energy the cell adhesion portion, where the cell adhesion-inhibitingmaterial is decomposed or denatured, and render the region notirradiated with energy the cell adhesion-inhibiting portion, where thecell adhesion-inhibiting material remains. At this time, the celladhesion portion contains the photocatalyst, a decomposition ordenaturation product of the cell adhesion-inhibiting material, and soon. The method for irradiating the energy, and others are the same as inthe first embodiment. Thus, description thereof is omitted.

In the embodiment also, it is allowable to cut a part of the patterningsubstrate where the cell adhesion portion and the celladhesion-inhibiting portion are formed in a pattern form, which may bereferred to as the cell culture patterning substrate hereinafter, stickthis part of the cell culture patterning substrate onto such as thebottom of a concave-form base material, and subsequently perform thecell-containing liquid applying process, which will be detailed later.

(3) Third Embodiment

The third embodiment of the present process is performed when thepatterning substrate is the third embodiment, and is a process ofirradiating energy from a given direction, thereby forming a patterncomposed of a cell adhesion-inhibiting portion where the cell adhesivematerial comprised in the cell adhesive layer is decomposed ordenatured, and a cell adhesion portion which is other than the celladhesion-inhibiting portion.

In the embodiment, energy is irradiated in a pattern form for formingthe cell adhesion-inhibiting portion, thereby exciting the photocatalystin the photocatalyst-containing layer so that the cell adhesive materialcan be decomposed or denatured. It is therefore possible to render theregion irradiated with energy the cell adhesion-inhibiting portion,where the cell adhesive material is decomposed or denatured, and renderthe region not irradiated with the energy the cell adhesion portion,where the cell adhesive material remains. At this time, the celladhesion-inhibiting portion comprises a small amount of the celladhesive material or comprises a decomposition product of the celladhesive material or the like, or the cell adhesive layer is completelydecomposed and removed to make the photocatalyst-containing layerexposed when the cell adhesive material is, for example, a materialwhich can be decomposed by action of the photocatalyst on the basis ofenergy irradiation. When the cell adhesive material is a material whichcan be denatured by action of the photocatalyst on the basis of energyirradiation, the cell adhesion-inhibiting portion comprises therein suchas a denaturation product thereof. The method for irradiating theenergy, and others are the same as in the first embodiment. Thus,description thereof is omitted herein.

In the embodiment also, it is allowable to cut a part of the patterningsubstrate where the cell adhesion portion and the celladhesion-inhibiting portion are formed in a pattern form, which may bereferred to as the cell culture patterning substrate hereinafter, stickthis part of the cell culture patterning substrate onto such as thebottom of a concave-form base material, and subsequently perform thecell-containing liquid applying process, which will be detailed later.

(4) Fourth Embodiment

The fourth embodiment of the present process is performed when thepatterning substrate is the fourth embodiment, and is a process ofirradiating energy from a given direction, thereby forming a patterncomposed of a cell adhesion portion where the cell adhesion-inhibitingmaterial comprised in the cell adhesion-inhibiting material layer isdecomposed or denatured and a cell adhesion-inhibiting portion which isother than the cell adhesion portion.

In the embodiment, energy is irradiated in a pattern form for formingthe cell adhesion portion, thereby exciting the photocatalyst in thephotocatalyst-containing layer so that the cell adhesion-inhibitingmaterial can be decomposed or denatured. It is therefore possible torender the region irradiated with energy the cell adhesion portion,where the cell adhesion-inhibiting material is decomposed or denatured,and render the region not irradiated with the energy the cell adhesionportion, where the cell adhesion-inhibiting material remains.

The wording “the cell adhesion-inhibiting material is decomposed ordenatured” means that the cell adhesion-inhibiting material is notcomprised, or the cell adhesion-inhibiting material is comprised in asmaller amount than the amount of the cell adhesion-inhibiting materialcomprised in the cell adhesion-inhibiting portion. When the celladhesion-inhibiting material is, for example, a material which can bedecomposed by action of the photocatalyst on the basis of energyirradiation, the cell adhesion portion comprises a small amount of thecell adhesion-inhibiting material, comprises a decomposition product orthe like of the cell adhesion-inhibiting material, the celladhesion-inhibiting material layer is completely decomposed and removedto make the photocatalyst-containing layer exposed, or the like. Whenthe cell adhesion-inhibiting material is a material which can bedenatured by action of the photocatalyst on the basis of energyirradiation, the cell adhesion portion comprises therein such as adenaturation product thereof. In the embodiment, the cell adhesionportion preferably comprises a cell adhesive material having celladhesive properties at least after the material is irradiated withenergy. This makes it possible to make the cell adhesive properties ofthe cell adhesion portion higher so that cells can be caused to adherehighly precisely only onto the cell adhesion portion. The method forirradiating the energy, and others are the same as in the firstembodiment. Thus, description thereof is omitted herein.

In the embodiment also, it is allowable to cut a part of the patterningsubstrate where the cell adhesion portion and the celladhesion-inhibiting portion are formed in a pattern form, which may bereferred to as the cell culture patterning substrate hereinafter, stickthis part of the cell culture patterning substrate onto such as thebottom of a concave-form base material, and subsequently perform thecell-containing liquid applying process, which will be detailed later.

(5) Fifth Embodiment

The fifth embodiment of the present process is performed when thepatterning substrate is the fifth embodiment, and is a process ofarranging the cell adhesive layer and a photocatalyst-containing layerside substrate having a base body and a photocatalyst-containing layercomprising a photocatalyst to dispose the cell adhesive layer and thephotocatalyst-containing layer facing each other, and then irradiatingenergy onto the resultant from a given direction, thereby forming apattern composed of a cell adhesion-inhibiting portion where the celladhesive material comprised in the cell adhesive layer is decomposed ordenatured, and a cell adhesion portion which is other than the celladhesion-inhibiting portion.

As shown in, for example, FIGS. 2A and 2B, in the energy irradiatingprocess of the embodiment, a cell adhesive layer 12 formed on a basematerial 1 and a photocatalyst-containing layer side substrate 23,having a photocatalyst-containing layer 21 comprising a photocatalystand a base body 22, are arranged to dispose the cell adhesive layer 12and the photocatalyst-containing layer 21 facing each other, and thenenergy 4 is irradiated, in a pattern form for forming celladhesion-inhibiting portions, onto the resultant through such as aphotomask 3 (FIG. 2A). According to this, regions where the celladhesive material is decomposed or denatured by the irradiation with theenergy are rendered cell adhesion-inhibiting portions 5, and regionswhere the cell adhesive material remains without being irradiated withthe energy are rendered cell adhesion portions 6 (FIG. 2B).

In the embodiment, the photocatalyst-containing layer side substrate isused to irradiate energy in a pattern form for forming the celladhesion-inhibiting portion, thereby exciting the photocatalyst in thephotocatalyst-containing layer so that the cell adhesive material can bedecomposed or denatured. It is therefore possible to render the regionirradiated with energy the cell adhesion-inhibiting portion, where thecell adhesive material is decomposed or denatured, and render the regionnot irradiated with the energy the cell adhesion portion, where the celladhesive material remains. At this time, the cell adhesion-inhibitingportion comprises a small amount of the cell adhesive material orcomprises a decomposition product or the like of the cell adhesivematerial, or the cell adhesive layer is completely decomposed andremoved to make the base material exposed when the cell adhesivematerial is, for example, a material which can be decomposed by actionof the photocatalyst on the basis of energy irradiation. When the celladhesive material is a material which can be denatured by action of thephotocatalyst on the basis of energy irradiation, the celladhesion-inhibiting portion comprises therein such as a denaturationproduct thereof.

The following will describe the photocatalyst-containing layer sidesubstrate used in the embodiment, which has a photocatalyst-containinglayer and a base body, and the method for irradiating the energy.

(a) Photocatalyst-Containing Layer Side Substrate

First, the photocatalyst-containing layer side substrate used in thepresent embodiment, which has a photocatalyst-containing layercomprising a photocatalyst, is described. The photocatalyst-containinglayer side substrate used in the embodiment is normally a substratehaving a photocatalyst-containing layer comprising a photocatalyst, andis usually a base body and substrate wherein a photocatalyst-containinglayer is formed on the base body. This photocatalyst-containing layerside substrate may have, for example, photocatalyst-containing layerside light-shielding portions formed in a pattern form, a primer layer,or the like. The following will describe each of the constituents of thephotocatalyst-containing layer side substrate used in the embodiment.

(i) Photocatalyst-Containing Layer

First, the photocatalyst-containing layer used in thephotocatalyst-containing layer side substrate is described. Thephotocatalyst-containing layer used in the embodiment is not limited toany special structure if the layer has a structure wherein thephotocatalyst in the layer can cause the decomposition or denaturationof the cell adhesive material in the adjacent or near cell adhesivelayer. The photocatalyst-containing layer may be made of a photocatalystand a binder, or may be made only of a photocatalyst.

The photocatalyst-containing layer used in the embodiment may be thephotocatalyst-containing layer 21 as illustrated in, for example, FIG.2A, which is formed on the whole of a surface of the base body 22; or aphotocatalyst-containing layer 21 as illustrated in, for example, FIG.3, which is formed in a pattern form on a base body 22.

In the case of forming the photocatalyst-containing layer in a patternform as described above, it is unnecessary that at the time ofirradiating energy to form the cell adhesion-inhibiting portion,patterning irradiation using such as a photomask is performed. Byfull-surface irradiation of the energy at this time, the celladhesion-inhibiting portion can be rendered a cell adhesion-inhibitingportion where the cell adhesive material comprised in the cell adhesivelayer is decomposed or denatured in a pattern form.

The method for patterning the photocatalyst-containing layer is notparticularly limited. For example, a method such as a photolithographymay be used.

The cell adhesive material only on the cell adhesive layer whichactually faces the photocatalyst-containing layer is decomposed ordenatured; therefore, the direction in which energy is irradiated may beany direction if the energy is irradiated onto the area where thephotocatalyst-containing layer and the cell adhesive layer face eachother. There is generated an advantage that the irradiated energy is notparticularly limited to energy composed of parallel constituents, suchas parallel light rays.

The photocatalyst-containing layer used in the embodiment may be thesame as described in the third embodiment in the above-mentioned item“1. Patterning Substrate Forming Process”. Thus, detailed descriptionthereof is omitted herein.

(ii) Base Body

The following will describe the base body used in thephotocatalyst-containing layer side substrate. Thephotocatalyst-containing layer side substrate is usually a member havingat least the base body and the photocatalyst-containing layer formed onthe base body. The material which constitutes the used base body isappropriately selected in accordance with the direction of the energyirradiation which will be detailed later, the matter as to whether ornot the pattern-formed body to be obtained needs transparency, and otherfactors.

The base body used in the present embodiment may be a member havingflexibility, such as a resin film, or may be a member having noflexibility, such as a glass plate. This is appropriately selected inaccordance with the method for the energy irradiation.

An anchor layer may be formed on the base body in order to improve theadhesive property between the base body surface and thephotocatalyst-containing layer. The anchor layer may be made of, forexample, a silane-based or titanium-based coupling agent.

(iii) Photocatalyst-Containing Layer Side Light-Shielding Portions

The photocatalyst-containing layer side substrate used in the presentembodiment may be a photocatalyst-containing layer side substrate onwhich photocatalyst-containing layer side light-shielding portions maybe formed into a pattern form. When the photocatalyst-containing layerside substrate having photocatalyst-containing layer sidelight-shielding portions is used in this way, it is unnecessary to use,at the time of irradiating energy, any photomask or to irradiate a laserray for drawing irradiation. It is therefore unnecessary to position aphotomask precisely onto the photocatalyst-containing layer sidesubstrate. Consequently, it is unnecessary to use any complicated stepor any expensive device for drawing irradiation, thereby producing anadvantage for costs.

Such a photocatalyst-containing layer side substrate havingphotocatalyst-containing layer side light-shielding portions can beclassified into the following two embodiments, depending on the positionwhere the photocatalyst-containing layer light-shielding portions areformed.

One of them is an embodiment as illustrated in, for example, FIG. 4,wherein photocatalyst-containing layer side light-shielding portions 24are formed on a base body 22 and further a photocatalyst-containinglayer 21 is formed on the photocatalyst-containing layer sidelight-shielding portions 24, thereby preparing aphotocatalyst-containing layer side substrate. The other is anembodiment as illustrated in, for example, FIG. 5, wherein aphotocatalyst-containing layer 21 is formed on a base body 22 andfurther photocatalyst-containing layer side light-shielding portions 24are formed thereon, thereby preparing a photocatalyst-containing layerside substrate.

In any one of the embodiments, the effect of energy-scattering in thebase body or the like can be made smaller than in the case of using aphotomask since the photocatalyst-containing layer side light-shieldingportions are arranged near the region where the photocatalyst-containinglayer and the cell adhesive layer are arranged. Accordingly, irradiationof energy in a pattern form can be extremely precisely attained.

In the present embodiment, an embodiment whereinphotocatalyst-containing layer side light-shielding portions 24 areformed on a photocatalyst-containing layer 21 as shown in FIG. 5 has anadvantage that at the time of arranging the photocatalyst-containinglayer and the cell adhesive layer in a predetermined position, the filmthickness of the light-shielding portions can be made consistent withthe width of the interval between the two layers, thereby using thelight-shielding portions as a spacer for making the interval constant.

In other words, when the photocatalyst-containing layer and the celladhesive material layer are arranged so as to be facing each other at agiven interval, the photocatalyst-containing layer side light-shieldingportions and the cell adhesive layer can be made closely adhesive toeach other, thereby making the dimension of the given interval precise.When the resultant in this state is irradiated with energy, celladhesion-inhibiting portions can be formed with a good precision sinceno cell adhesive material is decomposed or denatured in the celladhesive layer inside the region where the cell adhesive layer and thelight-shielding portions contact.

The method for forming the photocatalyst-containing layer sidelight-shielding portions is not specially limited, and is appropriatelyselected in accordance with properties of the face where thephotocatalyst-containing layer side light-shielding portions are to beformed, the performance of shielding required energy, and other factors.The method may be the same method for forming the light-shieldingportions, which are formed on the base material, described about thefirst embodiment in the item “1. Patterning Substrate Forming Process”.Thus, detailed description is omitted herein.

The above has described two cases, wherein the photocatalyst-containinglayer side light-shielding portions are formed between the base body andthe photocatalyst-containing layer and are formed on the surface of thephotocatalyst-containing layer. Besides, the photocatalyst-containinglayer side light-shielding portions may be formed on the base bodysurface on which no photocatalyst-containing layer is formed. In thisembodiment, for example, a photomask can be made close to this surfaceto such a degree that the photomask can be put on and taken off. Thus,this embodiment can be preferably used for the case that the pattern ofthe cell adhesion-inhibiting portions is varied for every small lot.

(iv) Primer Layer

The following will describe a primer layer used in thephotocatalyst-containing layer side substrate of the embodiment. Whenphotocatalyst-containing layer side light-shielding portions are formedinto a pattern form on a base body and a photocatalyst-containing layeris formed thereon so as to prepare a photocatalyst-containing layer sidesubstrate described above, a primer layer may be formed between thephotocatalyst-containing layer side light-shielding portions and thephotocatalyst-containing layer.

The effect and function of this primer layer are not necessarily clear,but would be as follows: the primer layer is formed between thephotocatalyst-containing layer side light-shielding portions and thephotocatalyst-containing layer, whereby the primer layer exhibits afunction of preventing the diffusion of impurities from openings whichare present in and between the light-shielding portions, the impuritiesbeing factors for blocking the decomposition or denaturation of the celladhesive material by action of the photocatalyst, in particular,residues generated when the photocatalyst-containing layer sidelight-shielding portions are patterned, or metal or metal ionimpurities. Accordingly, the formation of the primer layer makes itpossible that the processing of the decomposition or denaturation of thecell adhesive material advances with high sensitivity so as to yieldcell adhesion-inhibiting portions which are highly precisely formed.

The primer layer in the present embodiment is a layer for preventing theeffect of the photocatalyst from being affected by the impuritiespresent inside not only the photocatalyst-containing layer sidelight-shielding portions but also the openings made between thelight-shielding portions. It is therefore preferred to form the primerlayer over the entire surface of the light-shielding portions plus theopenings.

The primer layer in the present embodiment is not limited to any specialstructure if the primer layer is formed not to bring thephotocatalyst-containing layer side light-shielding portions and thephotocatalyst-containing layer of the photocatalyst-containing layerside substrate into contact with each other.

A material that forms the primer layer, though not particularlyrestricted, is preferably an inorganic material that is not likely to bedecomposed by action of the photocatalyst. Specifically, amorphoussilica can be cited. When such amorphous silica is used, a precursor ofthe amorphous silica is preferably a silicon compound that isrepresented by a general formula, SiX₄, X being halogen, methoxy group,ethoxy group, acetyl group or the like, silanol that is a hydrolysatethereof, or polysiloxane having an average molecular weight of 3000 orless.

A film thickness of the primer layer is preferably in the range of 0.001to 1 m and particularly preferably in the range of 0.001 to 0.1 μm.

b. Energy Irradiating Method

The following will describe the energy irradiating method in the presentembodiment. In the embodiment, the cell adhesive layer and thephotocatalyst-containing layer of the photocatalyst-containing layerside substrate are arranged at a given interval, and energy isirradiated onto the resultant.

The above-mentioned wording “arranging” means that the above-mentionedtwo layers; the photocatalyst-containing layer and the cell adhesivelayer are arranged in the state that the action of the photocatalyst cansubstantially work to the surface of the cell adhesive layer, andinclude not only the state that the two layers actually contact eachother, but also the state that the two layers are arranged at a giveninterval. The dimension of the interval is preferably 200 μm or less.

The dimension of the above-mentioned interval in the embodiment is morepreferably from 0.2 to 10 μm, even more preferably from 1 to 5 μm sincethe precision of the pattern to be obtained becomes very good andfurther the sensitivity of the photocatalyst becomes high so as to makegood the efficiency of the decomposition or denaturation of the celladhesive material in the cell adhesive layer. This range of the intervaldimension is particularly effective for the cell adhesive layer small inarea, which makes it possible to control the interval dimension with ahigh precision.

Meanwhile, in the case of treating the cell adhesive layer large inarea, for example, 300 mm or more×300 mm or more in size, it is verydifficult to make a fine interval as described above between thephotocatalyst-containing layer side substrate and the cell adhesivelayer without contacting each other. Accordingly, when the cell adhesivelayer has a relatively large area, the interval dimension is preferablyfrom 10 to 100 μm, more preferably from 50 to 75 μm. The limitation ofthe interval dimension into this range gives an advantageous effect thatthe cell adhesive material is not unevenly decomposed or denaturedwithout causing problems based on a fall in patterning precision, suchas a problem that a blurred pattern is obtained, or other problems, suchas a problem that the sensitivity of the photocatalyst deteriorates sothat the efficiency of decomposing or denaturing the cell adhesivematerial also deteriorates.

When energy is irradiated onto the cell adhesive material having arelatively large area as described above from an energy irradiatingdevice, a unit for positioning the photocatalyst-containing layer sidesubstrate and the cell adhesive layer inside the device is permitted toset the dimension of the interval therebetween preferably into the rangeof 10 to 200 μm, more preferably into the range of 25 to 75 μm. Thesetting of the interval dimension value into this range makes itpossible to arrange the photocatalyst-containing layer side substrateand the cell adhesive layer without causing a large drop in patterningprecision or in sensitivity of the photocatalyst, or bringing thesubstrate and the layer into contact with each other.

When the photocatalyst-containing layer and the surface of the celladhesive layer are arranged at a given interval as described above,active oxygen species generated from oxygen and water by action of thephotocatalyst can easily be released. In other words, if the intervalbetween the photocatalyst-containing layer and the cell adhesive layeris made narrower than the above-mentioned range, the active oxygenspecies are not easily released, so as to make the rate for decomposingor denaturing the cell adhesive material unfavorably small. If the twolayers are arranged at an interval larger than the above-mentionedrange, the generated active oxygen species do not reach the celladhesive layer easily. In this case also, the rate for decomposing ordenaturing the cell adhesive material becomes unfavorably small.

The method for arranging the photocatalyst-containing layer and the celladhesive layer to make such a very small interval evenly therebetweenis, for example, a method of using spacers. The use of the spacers inthis way makes it possible to make an even interval. In regions whichthe spacers contact, the action of the photocatalyst does not work ontothe surface of the cell adhesive layer; therefore, when the spacers arerendered spacers having a pattern similar to that of the cell adhesionportions, the cell adhesive material only inside regions where nospacers are formed can be decomposed or denatured so that highly precisecell adhesion-inhibiting portions can be formed. The use of the spacersalso makes it possible that the active oxygen species generated byaction of the photocatalyst reach the surface of the cell adhesivelayer, without diffusing, at a high concentration. Accordingly, highlyprecise cell adhesion-inhibiting portions can be effectively formed.

When the base material has one or more concave portions and the celladhesive layer is formed in the concave portion(s) as described above,energy may be irradiated onto the entire surface thereof by theabove-mentioned method. In the case of the plural concave portions, forexample, the exposure may be performed into the form of patternsdifferent from each other for the individual concave portions. Examplesof the method for performing the exposure into the form of patternsdifferent from each other for the individual concave portions asdescribed above include a method of arranging different masks for theindividual concave portions to irradiate energy by use of theabove-mentioned photocatalyst-containing layer side substrate; and amethod of arranging the photocatalyst-containing layer side substrateand a chromium mask, a stencil mask or the like at the tip of an opticalfiber to irradiate energy.

In order not to radiate any energy onto side walls of the concaveportions, the method for the exposure may be, for example, a method ofusing a cylindrical mask to irradiate energy only onto the bottom facesof the concave portions.

In the embodiment, it is sufficient that such arrangement state of thephotocatalyst-containing layer side substrate is maintained only duringthe irradiation of energy.

The energy irradiation (exposure) mentioned in the present embodiment isa concept that includes all energy line irradiation that can decomposeor denature the cell adhesive material by action of a photocatalyst onthe basis of energy irradiation, and is not restricted to lightirradiation.

The kind of the energy irradiated in the embodiment and related mattersthereof may be the same as described in the first embodiment. Thus,detailed description thereof is omitted.

The energy through the photomask in the embodiment may be irradiatedfrom either of the side of the base material and the side of thephotocatalyst-containing layer side substrate when the base material istransparent. On the other hand, when the base material is opaque, theenergy needs to be irradiated from the side of thephotocatalyst-containing layer side substrate.

In the embodiment also, it is allowable to cut a part of the patterningsubstrate where the cell adhesion portion and the celladhesion-inhibiting portion are formed in a pattern form, which may bereferred to as the cell culture patterning substrate hereinafter, stickthis part of the cell culture patterning substrate onto such as thebottom of a concave-form base material, and subsequently perform thecell-containing liquid applying process, which will be detailed later.

(6) Sixth Embodiment

The sixth embodiment of the present process is performed when thepatterning substrate is the sixth embodiment, and is a process ofarranging the cell adhesion-inhibiting material layer and aphotocatalyst-containing layer side substrate having a base body and aphotocatalyst-containing layer comprising a photocatalyst to dispose thecell adhesion-inhibiting material layer and the photocatalyst-containinglayer facing each other, and then irradiating energy onto the resultantfrom a given direction, thereby forming a pattern composed of a celladhesion portion where the cell adhesion-inhibiting material comprisedin the cell adhesion-inhibiting material layer is decomposed ordenatured, and a cell adhesion-inhibiting portion which is other thanthe cell adhesion portion.

In the embodiment, the photocatalyst-containing layer side substrate isused to irradiate energy in a pattern form for forming the cell adhesionportion, thereby exciting the photocatalyst in thephotocatalyst-containing layer so that the cell adhesion-inhibitingmaterial can be decomposed or denatured. It is therefore possible torender the region irradiated with energy the cell adhesion portion,where the cell adhesion-inhibiting material is decomposed or denatured,and render the region not irradiated with the energy the cell adhesionportion, where the cell adhesion-inhibiting material remains.

The wording “the cell adhesion-inhibiting material is decomposed ordenatured” means that the cell adhesion-inhibiting material is notcomprised, or the cell adhesion-inhibiting material is comprised in asmaller amount than the amount of the cell adhesion-inhibiting materialcomprised in the cell adhesion-inhibiting portion. When the celladhesion-inhibiting material is, for example, a material which can bedecomposed by action of the photocatalyst on the basis of energyirradiation, the cell adhesion portion comprises a small amount of thecell adhesion-inhibiting material or comprises a decomposition productof the cell adhesion-inhibiting material, the cell adhesion-inhibitingmaterial layer is completely decomposed and removed to make the basematerial exposed, or the like. When the cell adhesion-inhibitingmaterial is a material which can be denatured by action of thephotocatalyst on the basis of energy irradiation, the cell adhesionportion comprises therein such as a denaturation product thereof. In theembodiment, the cell adhesion portion preferably comprises a celladhesive material having cell adhesive properties at least after thematerial is irradiated with energy. This makes it possible to make thecell adhesive properties of the cell adhesion portion higher so thatcells are caused to adhere highly precisely only onto the cell adhesionportion. The method for irradiating the energy, and others are the sameas in the fifth embodiment. Thus, description thereof is omitted herein.

In the embodiment also, it is allowable to cut a part of the patterningsubstrate where the cell adhesion portion and the celladhesion-inhibiting portion are formed in a pattern form, which may bereferred to as the cell culture patterning substrate hereinafter, stickthis part of the cell culture patterning substrate onto such as thebottom of a concave-form base material, and subsequently perform thecell-containing liquid applying process, which will be detailed later.

3. Cell-Containing Liquid Applying Process

Lastly, the cell-containing liquid applying process in the method of theinvention for producing a cell culture substrate will be described. Thecell-containing liquid applying process is a process of applying acell-containing liquid onto the cell adhesion portion by aregion-selecting applying method of applying the cell-containing liquidselectively onto the patterned cell adhesion portion.

As described above, a cell-containing liquid is applied by theregion-selecting applying method in the invention; therefore, theresultant cell culture substrate can be rendered a highly precise cellculture substrate and further two or more kinds of cells can also becaused to adhere onto the substrate.

The following will describe each of the method and the elements in thepresent process.

(Region-Selecting Applying Method)

The region-selecting applying method in the invention is a methodcapable of causing a liquid to adhere selectively only onto one or moregiven regions. Examples of the method include jetting-out methods suchas ink-jetting method, printing methods such as letterpress printing,intaglio printing and gravure printing, and a pen point adhesion methodof absorbing a liquid into a convex tip by use of such as a capillaryphenomenon and bringing this tip into contact with a substrate to causethe liquid to adhere onto the substrate.

In the invention, it is preferred to use a jetting-out method such asink-jetting method. When this jetting-out method is used to apply acell-containing liquid onto the cell adhesion portion, a relativelylarge amount of the cell-containing liquid can be caused to adhere ontothe cell adhesion portion. In the invention, the use of ink-jettingmethod is more preferred. The use of the ink-jetting method makes itpossible to cause the cell-containing liquid to adhere very preciselyand effectively onto the cell adhesion portion. It is also possible toarrange plural kinds of cells in arbitrary positions by using pluralcell-containing liquids containing different kinds of cells and pluralink-jetting devices.

The ink-jetting method used in the invention may be any ink-jettingmethod that does not give damage to cells, and may be an ink-jettingmethod using any one selected from various manners such as a manner ofelectrifying a cell-containing liquid and jetting out this liquid whilecontrolling the liquid by a magnetic field, a manner of using apiezoelectric element to jet out an ink intermittently, a manner ofheating an ink and using bubbles therefrom to jet out the inkintermittently, an electrostatic jetting manner, an ultrasonic jettingmanner, a centrifugal jetting manner, and an inertia force head manner.In the invention, it is particularly preferred to use ink-jetting usinga piezoelectric element. Since the piezoelectric element using methoddoes not require the cell-containing liquid to be heated or otherprocesses, the method is preferred as a method for causing living cellsto adhere onto a substrate.

In the invention, it is preferred that the application of thecell-containing liquid by the region-selecting applying method isperformed in an atmosphere having a temperature or humidity at whichcells are not dried or annihilated. The atmosphere is appropriatelyselected in accordance with the kind of the cell-containing liquid whichwill be detailed later, and other factors. Usually, the temperature ispreferably from about 20 to 40° C., and the humidity is preferably fromabout 80 to 100%. In such an environment, microorganisms besides targetcells breed easily. It is therefore preferred to perform the applicationin a sterilized environment and give a sterilizing function to a deviceused in the region-selecting applying method.

(Cell-Containing Liquid)

The cell-containing liquid used in the embodiment is not limited to anyspecial kind if the liquid is a liquid which contains cells that can becaused to adhere onto the cell adhesion portion and cultured and whichis stable when the liquid is applied by the region-selecting applyingmethod. The cell-containing liquid is appropriately selected inaccordance with the kind of the region-selecting applying method andother factors.

The cell culture liquid may contain, for example, a culture solution, ora biological cell adhesive material besides cells. To protect the cells,the cells may be micro-encapsulated.

As a cell used in the present invention, except for, for instance,non-adhesive cells such as nervous tissue, liver, kidney, pancreas,blood vessel, brain, cartilage and blood corpuscle, all tissues presentin an organism and cells derived therefrom can be used. Furthermore,since even for generally non-adhesive cells, recently, in order toadhere and fix, a technology of modifying a cell membrane is devised;accordingly, as needs arise, the non-adhesive cells, when thistechnology is applied, can be used in the present invention.

The respective tissues such as mentioned above are formed of cellshaving various functions; accordingly, it is necessary to select desiredcells to use. For instance, in the case of the liver, it is formed of,other than hepatocytes, epithelial cells, endothelial cells, Kupffer'scells, fibroblasts, and fat-storing cells and the like. In this case,since the adhesiveness with a cell adhesive portion is differentdepending on the kinds of the cells, in accordance with a cell strain, acell adhesive material used in the cell adhesion portion and acomposition ratio thereof or the like have to be selected.

The cell-containing liquid used in the cell-containing liquid applyingprocess of the invention may be the same as described in the item“Ink-Jetting Cell-Containing Liquid”. Thus, detailed description isomitted herein.

It is preferred that the cell culture liquid is kept at a constanttemperature when the liquid is applied by the region-selecting applyingmethod or in the device for the method. This is because cells are easilyaffected by temperature and are annihilated outside a given temperaturerange.

(Cell Adhesion Portion)

The following will describe the cell adhesion portion onto which thecell-containing liquid is applied in the present process. In theinvention, the cell adhesion portion and the cell adhesion-inhibitingportion are formed in a pattern form in the above-mentioned energyirradiating process. It therefore becomes possible in the presentprocess to apply the cell-containing liquid only onto the cell adhesionportion without causing the cell-containing liquid to adhere onto thecell adhesion-inhibiting portion.

In the present process, the cell adhesion portion, which is irradiatedwith energy in the energy irradiating process, can be made hydrophilicand the cell adhesion-inhibiting portion can be made water repellent, inparticular, in the case of the second, fourth and sixth embodimentsdescribed about the above-mentioned patterning substrate formingprocess. Accordingly, when the cell-containing liquid is applied, thecell-containing liquid does not adhere onto the cell adhesion-inhibitingportion so that cells can be caused to adhere more highly precisely onlyonto the cell adhesion portion. According to a report, when cells arecaused to adhere onto the boundary region between property-differentregions of hydrophilic and water repellent regions, a form-change, suchas extension or orientation, is caused in accordance with the kind ofthe cells. Accordingly, in the above-mentioned embodiments, the form ofthe cells caused to adhere onto the cell adhesion portion is changed asthe case may be, so that the organization of the cells can be promoted.

It is preferred to keep the cell adhesion portion at a given temperaturewhen the cell-containing liquid is applied. This is based on thefollowing reason: in this case also, cells are easily affected bytemperature; therefore, the cells applied onto the cell adhesion portionare annihilated outside a given temperature range.

4. Others

If necessary, the method of the invention for producing a cell culturesubstrate may comprise a process other than the above-mentionedprocesses. Examples of the process include a pattern maintaining processof using the action of the photocatalyst to maintain the pattern.

In the case of the first, third and fifth embodiments described aboutthe above-mentioned pattern, the pattern maintaining process is aprocess of causing the cell-containing liquid onto the cell adhesionportion and then giving the action of the photocatalyst on the basis ofenergy irradiation to the cell adhesion-inhibiting portion, whereby thecells adhering onto the cell adhesion-inhibiting portion can be removedso that the pattern adhering onto the cell adhesion portion can behighly precisely maintained.

In the first and third embodiments, the pattern maintaining process canbe performed, for example, by irradiating energy onto the patterningsubstrate through a photomask having openings in the form of the patternof the above-mentioned cell adhesion-inhibiting portion(s). In the fifthembodiment, the pattern maintaining process can be performed bydisposing the cell adhesive layer and the photocatalyst-containing layerside substrate to face to each other and then irradiating energy ontothe patterning substrate through such as a photomask having openings inthe form of the pattern of the cell adhesion-inhibiting portion(s).

When the base material is transparent and one or more light-shieldingportions are formed in the form of the pattern of the above-mentionedcell adhesion portion(s) in anyone of the above-mentioned embodiments,the pattern maintaining process can be performed by irradiating energyonto the patterning substrate from the base material side thereofwithout using any photomask.

No special limitation is imposed to the energy irradiated at this timeif the energy is energy capable of removing the cells adhering onto thecell adhesion-inhibiting portion(s) by action of the photocatalyst onthe basis of the energy irradiation. The energy may be the same asirradiated in the above-mentioned energy irradiating process.

The pattern maintaining process may be performed immediately after thecell-containing liquid applying process of applying the cell-containingliquid on the cell adhesion portion(s). When the cells on the celladhesion portion(s) are cultured for a given period, the patternmaintaining process may be performed at an appropriate time inaccordance with the kind or the state of the cells in order not to causesuch an inconvenience that the cells adhere onto the celladhesion-inhibiting portion to make the pattern thick. The patternmaintaining process may be repeated.

B. Ink-Jetting Cell-Containing Liquid

The following will describe the ink-jetting cell-containing liquid(i.e., the cell-containing liquid for ink-jet). The cell-containingliquid can be classified into two embodiments. Each of the embodimentswill be described hereinafter.

1. First Embodiment

The first embodiment of the ink-jetting cell-containing liquid of theinvention is an ink-jetting cell-containing liquid which comprisescells, a culture solution and a biological cell adhesive material, andwhich is jet out in a pattern form onto a substrate by ink-jettingmethod, thereby forming a cell culture substrate onto which the cellsadhere in the pattern form.

In the embodiment, the ink-jetting cell-containing liquid comprisestherein the biological cell adhesive material; therefore, the adhesiveproperty between the cells jetted-out on the substrate and the substratecan be made good.

It is preferred to use the ink-jetting cell-containing liquid used inthe embodiment in, for example, the above-mentioned method for producinga cell culture substrate. This makes it possible such as that when thecells are cultured after the adhesion of the cells, the cells areregularly arranged or undergo a form-change so as to form tissues.

The following will describe each of the materials of the ink-jettingcell-containing liquid.

(Cells)

First, the cells used in the embodiment are described. The cells are notlimited to any special kind if the cells can be jetted out byink-jetting method and further the cells are stable when and after thecells are jetted.

The size of the cells that can be jetted out by ink-jetting method isusually from about 1 to 50 μm, preferably from about 10 to 20 μm.Examples of the cells having such a size include hepatocytes,Langerhans' islet cells, and vascular endothelial cells.

The content of such cells is appropriately selected in accordance withthe kind of the cells, the pore size of an ink-jetting device, or otherfactors. Usually, the content is preferably from 1×10⁵ to 1×10¹⁰cells/ml of the cell-containing liquid of the embodiment, morepreferably from 1×10⁶ to 1×10⁸ cells/ml thereof.

(Culture Solution)

The following will describe the culture solution used in the embodiment.The culture solution is a solution for culturing cells mentioned above.The above-mentioned cells can be stably present in an ink-jetting deviceor the like without being annihilated or the like.

The material used in this culture solution is preferably a materialwhich is stable after it is jetted out from an ink-jetting device.Examples thereof include mixtures of amino acids, vitamins, sugars, andsalt. In the embodiment, it is preferred to use a solution wherein thebiological cell adhesive material which will be detailed later or aserum is added to an Eagle's basal medium, a Fisher's medium or a ham'smedium, in which necessary amounts of the above-mentioned components aremixed beforehand, or an improved product thereof.

The content by percentage of the culture solution in the cell-containingliquid of the embodiment is appropriately selected in accordance withthe kind of the cells, or other factors. Usually, the content ispreferably from 0.5 to 2.0% by weight, more preferably from 1.0 to 1.5%by weight.

(Biological Cell Adhesive Material)

The following will describe the biological cell adhesive material usedin the embodiment. The biological cell adhesive material is a materialby which at the time of applying the ink-jetting cell-containing liquidof the embodiment onto a substrate or the like, the adhesive propertybetween the cells and the base material is made good.

The biological cell adhesive material is, for example, a material goodin adhesive properties to a specific kind of cells, or a material goodin adhesive properties to many kinds of cells. Specific examples thereofinclude fibronection, laminin, tenascin, vitronection, RGD(alginine-glycine-asparagic acid) sequence containing peptide, YIGSR(tyrosine-isoleucine-glycine-serine-arginine) sequence containingpeptide, collagen, atelocollagen, and gelatin. Of these, atelocollagenand gelatin are preferred from the viewpoint of the adjustment of theconcentration and viscosity of the cell-containing liquid.

Usually, the content by percentage of the biological cell adhesivematerial in the cell-containing liquid of the embodiment isappropriately selected in accordance with the kind of the cells, thekind of the biological cell adhesive material, and other factors, and ispreferably from 0.01 to 5.0% by weight, more preferably from 0.5 to 2.0%by weight.

2. Second Embodiment

The following will describe the second embodiment of the ink-jettingcell-containing liquid of the present invention. The ink-jettingcell-containing liquid of the embodiment is an ink-jettingcell-containing liquid which is jet out in a pattern form onto asubstrate by ink-jetting method, thereby forming a cell culturesubstrate onto which cells adhere in the pattern form, the cells beingmicro-encapsulated.

Since the used cells are micro-encapsulated in the embodiment, the cellsthemselves are not damaged when jetted out by ink-jetting method so thatthe survival rate of the jetted-out cells can be improved. It istherefore to improve the yield of the cell culture substrates obtainedby use of the ink-jetting cell-containing liquid.

It is preferred to use the ink-jetting cell-containing liquid used inthe embodiment in, for example, the above-mentioned method for producinga cell culture substrate. This makes it possible such as that when thecells are cultured after the adhesion of the cells, the cells areregularly arranged or undergo a form-change so as to form tissues.

The following will describe each of the materials of the ink-jettingcell-containing liquid.

(Cells)

The cells used in the embodiment are cells contained in themicrocapsules which will be detailed later. The cells are not limited toany special kind if the cells are stable in the microcapsules and arefurther stable when and after the cells are jetted from an ink-jettingdevice.

Examples of the cells include hepatocytes, Langerhans' islet cells,vascular endothelial cells, and cells which are poor in adhesiveproperty to a substrate and are easily damaged, such as brain cells andnerve cells.

The number of the cells contained in the microcapsules, which will bedetailed below, is usually from 1 to 100.

(Microcapsules)

The following will describe the microcapsules used in the embodiment. Nospecial limitation is imposed on the microcapsules if the microcapsulescontain the above-mentioned cells and protect the cells from impact,heat or the like when jetted out from an ink-jetting device. Themicrocapsules are, for example, microcapsules wherein thecell-containing liquid is contained in a capsule membrane made of a gelcompound such as gelatin. Such capsules can be formed, for example, bydropping down the material of the membrane and the cell-containingliquid from concentric nozzles. In general, such microcapsules have adiameter of about 100 μm to several millimeters, but the microcapsulesin the present manner desirably have a diameter of about 100 to 300 μm.

C. Ink-Jetting Device for Producing a Cell Culture Substrate

Lastly, the ink-jetting device of the invention for producing a cellculture substrate is described. The ink-jetting device is an ink-jettingdevice for producing a cell culture substrate comprising acell-containing liquid supplying section into which a cell-containingliquid is filled, a jetting-out section having a piezoelectricallydriving head unit for jetting out the cell-containing liquid suppliedfrom the cell-containing liquid supplying section onto a cell culturepatterning substrate, and a stage for fixing the cell culture patterningsubstrate, wherein the cell-containing liquid supplying section isprovided with a stirring means for dispersing cells homogeneously in thefilled cell-containing liquid, and the jetting-out section and stage areprovided with one or more temperature controlling means for keeping thetemperature of the cell-containing liquid constant before and after thejetting-out.

In the invention, the stirring means is arranged in the cell-containingliquid supplying section; therefore, cells can be made into the statethat the cells are homogeneously dispersed in the cell-containing liquidinside the supplying section. Accordingly, the content by percentage ofthe cells in the jetted-out cell-containing liquid can be made constantso that the number of the cells adhering onto the resultant cell culturesubstrate can be made even. This makes it possible to render the cellculture substrate a high-quality cell culture substrate. In theinvention, the ink-jetting device has the temperature controlling meansfor keeping the temperature of the jetting-out section and the stageconstant; therefore, it is possible to prevent the annihilation of thecells, for example, in the cell-containing liquid in the cell-containingliquid supplying section and in a channel extending from thiscell-containing liquid supplying section to orifices of thepiezoelectrically driving head unit, and in the cell-containing liquidjetted-out onto the cell culture patterning substrate fixed onto thestage, thereby improving the survival rate of the cells. Thus, the yieldof the resultant cell culture substrates can be improved.

As shown in, for example, FIG. 6, in an ink-jetting device of theinvention for producing a cell culture substrate, a cell-containingliquid is filled into a cell-containing liquid supplying section andthis cell-containing liquid supplying section 31 is provided with astirring means. The device also has a piezoelectrically driving headunit for jetting out the cell-containing liquid supplied from thiscell-containing liquid supplying section 31 onto a target cell culturepatterning substrate 30, and further has a jetting-out section 32 havinga temperature controlling means. Furthermore, a stage 33 onto which thecell culture patterning substrate 30 will be fixed is also provided witha temperature controlling means. In order to apply the cell-containingliquid onto a target location therein at this time, the stage 33 may bemoved or the jetting-out section 32 may be moved. Both of them may bemoved. The device may be provided with such as a control section 34 forcontrolling stirring in the cell-containing liquid supplying section 31and the temperature of the section 31 and controlling the temperature ofthe jetting-out section 32.

The cell-containing liquid used in the ink-jetting device of theinvention for producing a cell culture substrate is not limited to anyspecial kind if the liquid is stable when jetted out or stored in thecell-containing liquid supplying section. It is particularly preferredto use the cell-containing liquid described in the item “B. Ink-JettingCell-Containing Liquid”. This makes it possible to jet out cells stablyand improve the yield of the resultant cell culture substrates. The cellculture patterning substrate onto which the cell-containing liquid willbe jetted is not limited to any special kind if the substrate is asubstrate capable of culturing the jetted-out cells. In the invention,the substrate is in particular preferably a substrate having a cellculture patterning layer having a cell adhesion portion and a celladhesion-inhibiting portion as described in the item “A. Method forProducing a Cell Culture Substrate”. This makes it possible to arrangethe jetted-out cells along a pattern of the cell adhesion portion andthe cell adhesion-inhibiting portion or change the form of the cells soas to culture the cells into a tissue easily. It is preferred that onthe cell culture patterning substrate a position-detecting mark isformed at a given location therein. This makes the positioning of thecell culture patterning substrate easy, whereby the cell-containingliquid can be very precisely applied selectively only onto the celladhesion portion. No special limitation is imposed on theposition-detecting mark if the mark can be detected. Thus, the mark maybe such as the same mark as is ordinarily used for the positioning ofsubstrates.

The following will describe each of the constituents of the ink-jettingdevice of the invention for producing a cell culture substrate.

1. Cell-Containing Liquid Supplying Section

First, the cell-containing liquid supplying section of the ink-jettingdevice of the invention for producing a cell culture substrate isdescribed. The cell-containing liquid supplying section of theink-jetting device of the invention for producing a cell culturesubstrate is not limited to any special structure if the section is asection which can be filled with the cell-containing liquid and has afunction of stirring the cell-containing liquid.

The above-mentioned stirring function is any function that makes itpossible to disperse the filled cell-containing liquid homogeneously,without giving any damage resulting from the stirring to the cells inthe cell-containing liquid or lowering the activity of the cells, inorder to prevent the cells from being precipitated or annihilated in thecell-containing liquid. This function can be fulfilled by, for example,rotary stirring or vibratory stirring.

It is preferred that the cell-containing liquid supplying section iskept at a constant temperature. The temperature thereof may be adjustedwith a temperature adjusting means of the jetting-out section which willbe detailed below, or may be adjusted with a temperature adjusting meansset separately to the present section.

2. Jetting-Out Section

The following will describe the jetting-out section in the ink-jettingdevice of the invention for producing a cell culture substrate. Thepiezoelectrically driving unit in the ink-jetting device of theinvention for producing a cell culture substrate has a piezoelectricallydriving unit for jetting out the cell-containing liquid supplied fromthe cell-containing liquid supplying section onto the cell culturepatterning substrate through such as a hose, the temperature of whichcan be adjusted. This jetting-out section has a temperature controllingfunction.

In the invention, the width of openings in orifices of thepiezoelectrically driving head is preferably set into the range of 20 to500 μm. Preferably, the width is set into the range of about 20 to 250μm in the case of using the cell-containing liquid of an ordinary type,and is set into the range of about 100 to 500 μm in the case of theabove-mentioned microcapsules and the like. This makes it possible tosupply the cell-containing liquid stably without damaging the cells inthis liquid. The piezoelectrically driving head with orifices havingsuch an opening width may be equivalent to an ordinary piezoelectricallydriving head. Thus, detailed description thereof is omitted herein.

The temperature controlling means is not particularly limited if themeans makes it possible to adjust the temperature of such as the channelextending from the cell-containing liquid supplying section and thetemperature of the piezoelectrically driving head. Thus, the means maybe the same as is ordinarily used as a temperature controllingtemperature. Thus, description thereof is omitted herein.

3. Stage

The following will describe the stage. The stage which is used in theink-jetting device of the invention is not particularly limited if thestage makes it possible to fix the cell culture patterning substrate,onto which the cell-containing liquid will be jetted out, and has atemperature controlling means for keeping the temperature thereofconstant.

The temperature controlling means in the invention is not limited to anyspecial type or structure if the means makes it possible to keep thesubstrate jetted out from the jetting-out section at a targettemperature. Examples thereof include a means having a heating mechanismand a cooling mechanism for attaining cooling with, for example, wateror a Peltier element.

4. Others

If necessary, the ink-jetting device of the invention for producing acell culture substrate may appropriately have a member other than theabove-mentioned members. It is particularly preferred that the device isprovided with a humidity controlling means for controlling the humiditybetween the piezoelectrically driving head unit of the jetting-outsection and the cell culture patterning substrate. This makes itpossible to prevent cells from being dried to be annihilated between thepiezoelectrically driving head unit and the cell culture patterningsubstrate.

The humidity controlling means may be a means for adjusting the humiditylocally between the piezoelectrically driving head unit and the cellculture patterning substrate, or may be a means for adjusting thehumidity of the whole of the ink-jetting device of the invention forproducing a cell culture substrate.

It is preferred that the ink-jetting device of the invention is providedwith a carbon dioxide concentration controlling means for adjusting thecarbon dioxide concentration between the piezoelectrically driving headunit of the jetting-out section and the cell culture patterningsubstrate. The carbon dioxide concentration controlling means may be ameans for adjusting the carbon dioxide concentration locally between thepiezoelectrically driving head unit and the cell culture patterningsubstrate, or may be a means for adjusting the carbon dioxideconcentration in the whole of the ink-jetting device of the inventionfor producing a cell culture substrate.

The present invention is not limited to the above-mentioned embodiments.The embodiments are mere examples, and all techniques havingsubstantially the same technical concept and producing substantially thesame advantageous effects as the techniques recited in the claims of theinvention, and all equivalents thereof are included in the technicalscope of the invention.

EXAMPLES

Hereinafter, examples are shown and thereby the present invention willbe more specifically described.

Example 1

[Patterning Substrate Forming Process]

(Formation of a Patterning Substrate)

The following were mixed: 5.0 g of an organosilane, TSL 8114(manufactured by GE Toshiba Silicones), 0.55 g of a fluoroalkylsilane,TSL 8233 (manufactured by GE Toshiba Silicones), and 2.36 g of 0.005 Nhydrochloric acid. The mixture was mixed 12 hours while stirred. Thissolution was diluted 100 times with isopropyl alcohol, and further thediluted solution was applied onto a soda glass substrate subjectedbeforehand to alkali treatment by spin coating. The substrate was driedat a temperature of 150° C. for 10 minutes, thereby forming a patterningsubstrate having a cell adhesion-inhibiting material layer, theproperties of which were able to be changed from celladhesion-inhibiting properties to cell adhesive properties bydecomposition reaction based on a photocatalyst.

[Energy Irradiating Process]

(Formation of a Photomask Having a Photocatalyst-Containing Layer)

The following were mixed: 3 g of isopropyl alcohol, 0.4 g of anorganosilane, TSL 8114 (manufactured by GE Toshiba Silicones), and 1.5 gof a photocatalyst inorganic coating agent, ST-K01 (manufactured byISHIHARA SANGYO KAISHA, LTD). The mixture was then heated at 100° C. for20 minutes while stirred.

This solution was diluted 10 times with isopropyl alcohol, and furtherthe diluted solution was applied onto a quartz photomask substratesubjected beforehand to alkali treatment, on which a stripe patterncomposed of light-shielding portions 500 μm in width and space portions150 μm in width was formed, by spin coating. The substrate was dried ata temperature of 150° C. for 10 minutes to advance hydrolysis andpolycondensation reaction, thereby forming a photomask having aphotocatalyst-containing layer, 0.2 μm in film thickness, wherein thephotocatalyst was strongly fixed onto an organopolysiloxane.

(Energy Irradiation)

Ultraviolet rays were irradiated from a mercury lamp through theabove-mentioned photomask onto the above-mentioned patterning substrateat an energy amount of 6 J/cm² to yield a cell culture patterningsubstrate having a cell adhesive surface patterned so as to haveunexposed portions having cell adhesion-inhibiting properties andexposed portions having cell adhesive properties.

[Cell-Containing Liquid Applying Process]

(Preparation of a Cell-Containing Liquid)

The steps for preparing cell-containing liquids originating from varioustissues are described in detail in, for example, “Soshikibaiyo noGijyutsu, Dai San Han, Kiso”, edited by The Japanese Tissue CultureAssociation and published by Asakura Shoten, and other documents. In thepresent process, primary human umbilical cord vein endothelial cells(HUVEC) were obtained by the method described on and after page 146 ofthis document.

(Application of the Cell-Containing Liquid)

An ink-jetting device (orifice width: 75 μm), a temperature and humidityadjusting device and a cell-containing liquid stirring device werefitted to an XY stage dispenser device manufactured by Sony Corporation,and then the cell-containing liquid was stirred therein at a temperatureof 37° C.

The cell-containing liquid was inoculated onto the cell adhesionportions of the cell culture patterning substrate under the followingenvironment conditions: a temperature of 37° C., a ratio by volume of 5%of carbon dioxide, a ratio by volume of 95% of air, and a relativehumidity of 99%. The cell culture patterning substrate was then left asit was, so as to stand still for 2 hours. Next, this cell culturesubstrate was quietly arranged inside a culture dish, and a DMEM mediumto which 10% fetal calf serum was added was attentively and quietlypoured into the dish so as to culture the cells at 37° C. and a relativehumidity of 90% in the environment of 5% by volume of carbon dioxide and95% by volume of air for 16 hours.

The cells adhering onto the substrate was observed to recognize that thecells were oriented in the directions along all regions where the cellswere cultured and the cells had an extended/developed shape.

The DMEM medium was exchanged with a DMEM medium to which 10 ng/ml ofbFGF (manufactured by Sigma) was added. The culture of the cells wascontinued at 37° C. in the environment of 5% by volume carbon dioxidefor 24 hours. It was observed that the cells formed a continuouscapillary tissue.

Comparative Example

A plasma-treated culture dish was used instead of the cell culturepatterning substrate to perform the same experiment as in Example 1. Asa result, the cells adhered in a pattern form onto the dish but thecells were not oriented or extended/developed. Furthermore, even if thebFGF was added to the medium, a continuous capillary was not formed, andsmall tissue slices were formed at random.

Example 2

[Patterning Substrate Forming Process, and Energy Irradiating Process]

The same patterning substrate forming process and energy irradiatingprocess as in Example 1 were performed except that an array-formphotomask having openings, 1000 μm in size, arranged at intervals of2000 μm was used, thereby forming a cell culture patterning substrate.

[Cell-Containing Liquid Applying Process]

(Preparation of Cell-Containing Microcapsules)

A cell-containing liquid was prepared in the same steps as in Example 1.Subsequently, a 40% solution of gelatin succinate (manufactured by NippiInc.) in pure water was stirred at 50° C. for 20 minutes. This gelatinsolution and the previously-prepared cell-containing liquid were droppeddown from coaxial nozzles having inside diameters of 300 μm and 200 μm,respectively, onto a Waymouth MB 752/1 medium solution, the temperatureof which was heated to 25° C., thereby preparing microcapsulescontaining the cells.

(Application of the Cell-Containing Liquid)

In the same way as in Example 1, the cell-containing microcapsulesolution was dropped down from an ink-jetting device, onto which ajetting-out head having orifices of 500 μm width was set, onto the celladhesion portions of the cell culture patterning substrate, therebypatterning the cells. This substrate was allowed to stand still in anincubator 37° C. in temperature, to which CO₂ was added to give a ratioby volume of 5%, for 3 hours, thereby causing hepatocytes to adhere ontothe substrate.

Next, the cell culture substrate onto which the cells adhered wasimmersed into a culture dish filled with a medium solution, and theculture of the cells was continued for 48 hours while the mediumsolution was exchanged. The cells were then observed with an opticalmicroscope. As a result, it was recognized that the cells adhered in anarray form along the cell adhesion portions on the cell culturepatterning substrate.

Example 3

[Patterning Substrate Forming Process]

The following were mixed: 3 g of isopropyl alcohol, 0.4 g of anorganosilane, TSL 8114 (manufactured by GE Toshiba Silicones), 0.04 g ofN-(2-aminoethyl)-3-aminopropyltrimethoxysilane (manufactured by HuelsAmerica Inc.), and 1.5 g of a photocatalyst inorganic coating agent,ST-K01 (manufactured by ISHIHARA SANGYO KAISHA, LTD). The mixture wasthen heated at 100° C. for 20 minutes while stirred.

This solution was applied onto a quartz glass substrate subjectedbeforehand to alkali treatment by spin coating, and the substrate wasdried at a temperature of 150° C. for 10 minutes to advance hydrolysisand polycondensation reaction, thereby forming a patterning substratehaving a photocatalyst-containing cell adhesive layer, 0.2 μm in filmthickness, containing the photocatalyst fixed strongly onto anorganopolysiloxane and having properties changeable from cell adhesiveproperties to cell adhesion-inhibiting properties by action of thephotocatalyst on the basis of energy irradiation.

[Energy Irradiating Process]

Ultraviolet rays were irradiated from a mercury lamp (wavelength: 365nm) through a photomask onto this patterning substrate at an illuminanceof 300 mW/cm² for 900 seconds to yield a cell culture patterningsubstrate wherein the resultant unexposed portions were cell adhesionportions and the resultant exposed portions were celladhesion-inhibiting portions.

[Cell-Containing Liquid Applying Process]

A cell-containing liquid was applied onto the cell culture patterningsubstrate in the same way as in the cell-containing liquid applyingprocess in Example 1. The cells adhering onto the substrate were thenobserved to recognize that the cells were oriented in the directionsalong all regions where the cells were cultured and the cell had anextended/developed shape.

The DMEM medium was exchanged with a DMEM medium to which 10 ng/ml ofbFGF (manufactured by Sigma) was added. The culture of the cells wascontinued at 37° C. in the environment of 5% by volume carbon dioxidefor 24 hours. It was observed that the cells formed a continuouscapillary tissue.

Example 4

[Patterning Substrate Forming Process]

The following were mixed: 3 g of isopropyl alcohol, 0.4 g of anorganosilane, TSL 8114 (manufactured by GE Toshiba Silicones), 0.04 g ofa fluoroalkylsilane, TSL 8233 (manufactured by GE Toshiba Silicones),and 1.5 g of a photocatalyst inorganic coating agent, ST-K01(manufactured by ISHIHARA SANGYO KAISHA, LTD). The mixture was thenheated at 100° C. for 20 minutes while stirred.

This solution was applied onto a quartz substrate, on the rear face ofwhich a light-shielding pattern composed of light-shielding portions of300 μm width and opening portions of 60 μm width was formed, by spincoating. The substrate was dried at a temperature of 150° C. for 10minutes to advance hydrolysis and polycondensation reaction, therebyforming a patterning substrate having a photocatalyst-containing celladhesion-inhibiting material layer, 0.2 μm in film thickness, containingthe photocatalyst fixed strongly onto an organopolysiloxane and havingproperties changeable from cell adhesion-inhibiting properties to celladhesion properties by action of the photocatalyst on the basis ofenergy irradiation.

[Energy Irradiating Process]

Ultraviolet rays were irradiated from a mercury lamp (wavelength: 254nm) onto the above-mentioned patterning substrate from the rear faceside thereof at an exposure quantity of 5 J/cm² to yield a cell culturepatterning substrate having a pattern wherein the resultant unexposedportions were cell adhesion-inhibiting portions and the resultantexposed portions were cell adhesion portions.

[Cell-Containing Liquid Applying Process]

A cell-containing liquid was applied onto the cell culture patterningsubstrate in the same way as in the cell-containing liquid applyingprocess in Example 1. The cells adhering onto the substrate were thenobserved to recognize that the cells were oriented in the directionsalong all regions where the cells were cultured and the cell had anextended/developed shape.

The DMEM medium was exchanged with a DMEM medium to which 10 ng/ml ofbFGF (manufactured by Sigma) was added. The culture of the cells wascontinued at 37° C. in the environment of 5% by volume carbon dioxidefor 24 hours. It was observed that the cells formed a continuouscapillary tissue.

Example 5

[Patterning Substrate Forming Process]

(Formation of a Photocatalyst-Containing Layer)

The following were mixed: 3 g of isopropyl alcohol, 0.4 g of anorganosilane, TSL 8114 (manufactured by GE Toshiba Silicones), and 1.5 gof a photocatalyst inorganic coating agent, ST-K01 (manufactured byISHIHARA SANGYO KAISHA, LTD). The mixture was then heated at 100° C. for20 minutes while stirred.

This solution was applied onto a quartz glass substrate subjectedbeforehand to alkali treatment by spin coating, and the substrate wasdried at a temperature of 150° C. for 10 minutes to advance hydrolysisand polycondensation reaction, thereby forming, on the substrate, aphotocatalyst-containing layer, 0.2 μm in film thickness, wherein thephotocatalyst was fixed strongly onto an organopolysiloxane.

(Formation of a Cell Adhesive Layer)

There were mixed 0.2 mg of fibronection, F-4759 (manufactured by Sigma)and 200 ml of pure water, and this aqueous solution was dropped downonto the above-mentioned photocatalyst-containing layer, which wasformed on the substrate, at a rate of 300 μl per cm² of the area of thesubstrate. This was allowed to stand still at 4° C. for 24 hours.Furthermore, the substrate washed 2 times with PBS, and exposed tonitrogen gas so as to be dried, thereby yielding a patterning substratehaving, on the substrate, the photocatalyst-containing layer and a celladhesive layer.

[Energy Irradiating Process]

Subsequently, ultraviolet rays were irradiated from a mercury lamp,through a stripe-form photomask having light-shielding portions of 80 μmwidth and space portions of 300 μm width, onto this patterning substrateat 5 J/cm² (wavelength: 254 nm) to yield a cell culture patterningsubstrate having the cell adhesion layer patterned to have unexposedportions having cell adhesive properties and exposed portions havingcell adhesion-inhibiting properties.

[Cell-Containing Liquid Applying Process]

A cell-containing liquid was applied onto the cell culture patterningsubstrate in the same way as in the cell-containing liquid applyingprocess in Example 1. The cells adhering onto the substrate were thenobserved to recognize that the cells were oriented in the directionsalong all regions where the cells were cultured and the cell had anextended/developed shape.

The DMEM medium was exchanged with a DMEM medium to which 10 ng/ml ofbFGF (manufactured by Sigma) was added. The culture of the cells wascontinued at 37° C. in the environment of 5% by volume carbon dioxidefor 24 hours. It was observed that the cells formed a continuouscapillary tissue.

Example 6

[Patterning Substrate Forming Process]

(Formation of a Photocatalyst-Containing Layer)

The following were mixed: 3 g of isopropyl alcohol, 0.4 g of anorganosilane, TSL 8114 (manufactured by GE Toshiba Silicones), and 1.5 gof a photocatalyst inorganic coating agent, ST-K01 (manufactured byISHIHARA SANGYO KAISHA, LTD). The mixture was then heated at 100° C. for20 minutes while stirred.

This solution was applied onto a quartz glass substrate subjectedbeforehand to alkali treatment by spin coating, and the substrate wasdried at a temperature of 150° C. for 10 minutes to advance hydrolysisand polycondensation reaction, thereby forming, on the substrate, aphotocatalyst-containing layer, 0.2 μm in film thickness, wherein thephotocatalyst was fixed strongly onto an organopolysiloxane.

(Formation of a Cell Adhesion-Inhibiting Material Layer)

A solution composed of 5 g of isopropyl alcohol, 0.4 g of anorganosilane, TSL 8114 (manufactured by GE Toshiba Silicones), and 0.04g of a fluoroalkylsilane, TSL 8233 (manufactured by GE ToshibaSilicones) was applied onto this substrate by spin coating, and then thesubstrate was dried at 150° C. for 10 minutes, thereby forming a celladhesion-inhibiting material layer.

[Energy Irradiating Process]

Ultraviolet rays were irradiated from a mercury lamp through a photomaskonto this patterning substrate at 6 J/cm² (wavelength: 254 nm) to yielda cell culture patterning substrate having a cell adhesive surfacepatterned so as to have unexposed portions having celladhesion-inhibiting properties and exposed portions having cell adhesiveproperties.

[Cell-Containing Liquid Applying Process]

A cell-containing liquid was applied onto the cell culture patterningsubstrate in the same way as in the cell-containing liquid applyingprocess in Example 1. The cells adhering onto the substrate were thenobserved to recognize that the cells were oriented in the directionsalong all regions where the cells were cultured and the cell had anextended/developed shape.

The DMEM medium was exchanged with a DMEM medium to which 10 ng/ml ofbFGF (manufactured by Sigma) was added. The culture of the cells wascontinued at 37° C. in the environment of 5% by volume carbon dioxidefor 24 hours. It was observed that the cells formed a continuouscapillary tissue.

Example 7

[Patterning Substrate Forming Process]

The following were mixed: 3 g of isopropyl alcohol, 0.4 g of anorganosilane, TSL 8114 (manufactured by GE Toshiba Silicones), and 0.4 gof aminopropyltriethoxysilane. The mixture was then heated at 100° C.for 20 minutes while stirred. This solution was applied onto a quartzglass substrate subjected beforehand to alkali treatment by spincoating. The substrate was dried at a temperature of 150° C. for 10minutes to advance hydrolysis and polycondensation reaction, therebyforming an organopolysiloxane layer, about 80 nm in film thickness,containing amino groups, on the substrate. In this way, a patterningsubstrate was formed.

[Energy Irradiating Process]

Energy irradiation was performed using a photomask having aphotocatalyst-containing layer in the same way as in Example 1. Thisgave a cell culture patterning substrate having a cell adhesive surfacepatterned so as to have unexposed portions having cell adhesionproperties and exposed portions having cell adhesion-inhibitingproperties.

[Cell-Containing Liquid Applying Process]

A cell-containing liquid was applied onto the cell culture patterningsubstrate in the same way as in the cell-containing liquid applyingprocess in Example 1. The cells adhering onto the substrate were thenobserved to recognize that the cells were oriented in the directionsalong all regions where the cells were cultured and the cell had anextended/developed shape.

The DMEM medium was exchanged with a DMEM medium to which 10 ng/ml ofbFGF (manufactured by Sigma) was added. The culture of the cells wascontinued at 37° C. in the environment of 5% by volume carbon dioxidefor 24 hours. It was observed that the cells formed a continuouscapillary tissue.

Example 8

[Patterning Substrate Forming Process, and Energy Irradiating Process]

The following were mixed: 3 g of isopropyl alcohol, 0.2 g of anorganosilane, TSL8114 (manufactured by GE Toshiba Silicones), and 0.2 gof PEG-silane (Methoxypolyethyleneglycol 5,000 trimethylsilyl ether,Fluka). The mixture was heated at 100° C. for 20 minutes while stirred.This solution was applied onto a washed glass substrate (thickness:about 0.1 mm) by spin coating. Subsequently, the substrate was subjectedto heating treatment at 150° C. for 10 minutes, thereby forming, on thesubstrate, a cell adhesion-inhibiting material layer, 160 nm in filmthickness, made of an organopolysiloxane layer containing polyethyleneglycol. In this way, a patterning substrate was yielded.

Next, the same energy irradiating process as in Example 1 was performedto form a cell culture patterning substrate. This cell culturepatterning substrate was cut into a 21 mm square. Subsequently, a holeof 14 mm in diameter was made in the center of the bottom face of acommercially available plastic dish (manufactured by Corning Inc.) 35 mmin diameter. The glass substrate of the cut cell culture patterningsubstrate was stuck onto the plastic dish with an adhesive agent, KE45T(manufactured by Shin-Etsu Chemical Co. Ltd).

[Cell-Containing Liquid Applying Process]

The plastic dish was sterilized with a germicidal lamp, washed with PBS,and then washed with a DMEM medium. Thereafter, a cell-containing liquidwas applied onto the dish and cells were cultured in the same way as inExample 1 except that no cell culture substrate was arranged in thedish. As a result, it was recognized that the cells were oriented alongthe cell adhesion portions inside the plastic dish and the cells had anextended/developed shape.

Example 9

A quartz substrate of about 0.1 mm thickness was used to form a cellculture patterning substrate and cut this cell culture patterningsubstrate into a 21 mm square in the same way as in Example 2.Thereafter, in the same way as in Example 8, the quartz substrate of thecut cell culture patterning substrate was stuck onto the same plasticdish as described above.

[Cell-Containing Liquid Applying Process]

A cell-containing liquid was applied into a plastic dish and cells werecultured in the same way as in Example 8. As a result, it was recognizedthat the cells were oriented along the cell adhesion portions inside theplastic dish and the cells had an extended/developed shape.

Example 10

A quartz substrate of about 0.1 mm thickness was used to form a cellculture patterning substrate and cut this cell culture patterningsubstrate into a 21 mm square in the same way as in Example 3.Thereafter, in the same way as in Example 8, the quartz substrate of thecut cell culture patterning substrate was stuck onto the same plasticdish as described above.

[Cell-Containing Liquid Applying Process]

A cell-containing liquid was applied into a plastic dish and cells werecultured in the same way as in Example 8. As a result, it was recognizedthat the cells were oriented along the cell adhesion portions inside theplastic dish and the cells had an extended/developed shape.

Example 11

A quartz substrate of about 0.1 mm thickness was used to form a cellculture patterning substrate and cut this cell culture patterningsubstrate into a 21 mm square in the same way as in Example 4.Thereafter, in the same way as in Example 8, the quartz substrate of thecut cell culture patterning substrate was stuck onto the same plasticdish as described above.

[Cell-Containing Liquid Applying Process]

A cell-containing liquid was applied into aplastic dish and cells werecultured in the same way as in Example 8. As a result, it was recognizedthat the cells were oriented along the cell adhesion portions inside theplastic dish and the cells had an extended/developed shape.

Example 12

A quartz substrate of about 0.1 mm thickness was used to form a cellculture patterning substrate and cut this cell culture patterningsubstrate into a 21 mm square in the same way as in Example 5.Thereafter, in the same way as in Example 8, the quartz substrate of thecut cell culture patterning substrate was stuck onto the same plasticdish as described above.

[Cell-Containing Liquid Applying Process]

A cell-containing liquid was applied into a plastic dish and cells werecultured in the same way as in Example 8. As a result, it was recognizedthat the cells were oriented along the cell adhesion portions inside theplastic dish and the cells had an extended/developed shape.

Example 13

A quartz substrate of about 0.1 mm thickness was used to form a cellculture patterning substrate and cut this cell culture patterningsubstrate into a 21 mm square in the same way as in Example 6.Thereafter, in the same way as in Example 8, the quartz substrate of thecut cell culture patterning substrate was stuck onto the same plasticdish as described above.

[Cell-Containing Liquid Applying Process]

A cell-containing liquid was applied into a plastic dish and cells werecultured in the same way as in Example 8. As a result, it was recognizedthat the cells were oriented along the cell adhesion portions inside theplastic dish and the cells had an extended/developed shape.

Example 14

A quartz substrate of about 0.1 mm thickness was used to form a cellculture patterning substrate and cut this cell culture patterningsubstrate into a 21 mm square in the same way as in Example 7.Thereafter, in the same way as in Example 8, the quartz substrate of thecut cell culture patterning substrate was stuck onto the same plasticdish as described above.

[Cell-Containing Liquid Applying Process]

A cell-containing liquid was applied into aplastic dish and cells werecultured in the same way as in Example 8. As a result, it was recognizedthat the cells were oriented along the cell adhesion portions inside theplastic dish and the cells had an extended/developed shape.

1-16. (canceled)
 17. The method for producing a cell culture substrateaccording to claim 1, wherein the region-selecting applying method is anink-jetting method.
 18. An ink-jetting cell-containing liquid comprisingcells, a culture solution and a biological cell adhesive material, andjets out in a pattern form onto a substrate by an ink-jetting method,thereby forming a cell culture substrate onto which the cells adhere inthe pattern form.
 19. An ink-jetting cell-containing liquid jets out ina pattern form onto a substrate by an ink-jetting method to form a cellculture substrate onto which cells adhere in the pattern form, and thecells being micro-encapsulated.
 20. The ink-jetting cell-containingliquid according to claim 18, which is the cell-containing liquid usedin the method for producing a cell culture substrate according to claim17.
 21. The ink-jetting cell-containing liquid according to claim 19,which is the cell-containing liquid used in the method for producing acell culture substrate according to claim
 17. 22. An ink-jetting devicefor producing a cell culture substrate, comprising a cell-containingliquid supplying section into which a cell-containing liquid is filled,a jetting-out section having a piezoelectrically driving head unit forjetting out the cell-containing liquid supplied from the cell-containingliquid supplying section onto a cell culture patterning substrate, and astage for fixing the cell culture patterning substrate, wherein thecell-containing liquid supplying section is provided with a stirringmeans for dispersing cells homogeneously in the cell-containing liquidfilled, and the jetting-out section and the stage are provided with oneor more temperature controlling means for keeping a temperature of thecell-containing liquid constant before and after a jetting-out.
 23. Theink-jetting device for producing a cell culture substrate according toclaim 22, which is provided with a humidity controlling means forcontrolling a humidity between the piezoelectrically driving head unitand the cell culture patterning substrate fixed onto the stage.